Growth factor antagonists for organ transplant alloimmunity and arteriosclerosis

ABSTRACT

The present invention provides materials and methods for antagonizing the function of vascular endothelial growth factor receptors, platelet derived growth factor receptors and other receptors, to prevent, inhibit, or ameliorate allograft rejection or arteriosclerosis in organisms that receive an organ transplant.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority of U.S.Provisional Application No. 60/888,067, filed Feb. 2, 2007, and U.S.Provisonal Application No. 60/888,305, filed Feb. 5, 2007. Thedisclosure of each priority application is incorporated herein byreference in its entirety.

BACKGROUND

Interaction of innate and adaptive immunity leads to alloimmuneresponses that may be detrimental to cardiac allografts and hearttransplant recipients. Antigen-presenting cells (APC) initiateallorecognition by processing foreign peptides, migrating to secondarylymphoid tissue, and presenting these peptides to recipient lymphocytes.After recognition, alloreactive T lymphocytes proliferate and migrate totheir target tissue. Although the current immunosuppressive regimentseffectively inhibit the proliferation of alloreactive T lymphocytes,they have several metabolic, infectious, renal and malignantside-effects. In addition, the long-term survival of heart transplantpatients is decreased by gradual concentric intimal thickening of largeand small allograft coronary arteries—cardiac allograftarteriosclerosis—despite the use of modern immunosuppression.

The lymphatic network forms a conduct system that transfers interstitialfluids and inflammatory cells from the target tissue to secondary LN,and is essential in the activation of adaptive immunity. Vascularendothelial growth factor C (VEGF-C) and its receptor VEGFR-3 are thekey regulators for lymphatic growth. VEGF-C is essential in thedevelopment and maintenance of the lymphatic system, and improperlymphangiogenesis is related to many pathological conditions. Lymphaticvascular insufficiency leads to lymphedema, whereas extensivelymphangiogenesis is often seen in tumor metastasis and inflammatorysituations. During inflammation, macrophages are a rich source forVEGF-C, and pro-inflammatory cytokines such as TNF-α, IL-1α and -β (15)as well as TGF-β (16.) induce VEGF-C expression. Dendritic cells (DC)may express VEGFR-3 during inflammation (Hamrah et al., (2003), Am J.Pathol., 163: 57-6817) which renders them responsive for VEGF-C-inducedmigration (Chen et al., (2004), Nat. Med., 10: 813-81518). Also,lymphatic endothelial cells (EC)—in contrast to vascular EC—secreteCCL21 chemokine that mediates CCR7+ inflammatory cell traffic tolymphoid organs and peripheral effector sites. (See Kriehuber et al.,(2001), J. Exp. Med., 194: 797-808; Saeki et al., (1999), J. Immunol.162: 2472-2475; Campbell et al., (1998), J. Cell. Biol. 141: 1053-1059;and Lo et al., (2003), J. Clin. Invest. 112: 1495-1505.

Corneal transplant is currently the most successful tissuetransplantation procedures in humans, with a first year survival rate ashigh as 90%, even in the absence of routine HLA tying and with minimalimmunosuppressive therapy. The healthy cornea is generally anon-vascular tissue. DeVries, U.S. Patent Publication No. 2003/0180294purports to describe use of a VEGFR-3 inhibitor to reducelymhangiogenesis in a transplanted cornea to extend its survival. Chenet al., (2004), Nat. Med., 10: 813-81518, purport to describe thatblockade of VEGFR-3 in corneal transplants suppresses corneal antigenpresenting dendritic cells, delaying rejection of corneal transplants.The same research group previously reported that dendritic cells in thecornea were VEGFR-3⁺, whereas similar dendritic cells were absent in theskin, even though the cornea shares embryological origins with the skin.

A need exists for all transplanted tissues and organs, especiallyvascularized tissues and organs, for new materials and methods forslowing, reducing, or eliminating rejection and also for slowing,reducing, or eliminating graft arteriosclerosis.

SUMMARY OF THE INVENTION

The present invention provides materials and methods to improve theoutcomes of transplant recipients, e.g., by postponing or inhibiting orreducing or ameliorating an immune reaction against the transplant(rejection), and/or by postponing or inhibiting or reducing orameliorating arteriosclerosis or other deleterious side effects oftenassociated with transplants.

Thus, in one embodiment, the invention is a method for inducingtolerance or inhibiting rejection of a cell, tissue, or organtransplant, or for inhibiting arteriosclerosis in a transplantrecipient. For example, one such method comprises administering to amammalian transplant recipient a composition that comprises a growthfactor inhibitor, such as an endothelial growth factor inhibitor, in anamount effective to induce tolerance for the transplant by therecipient, or inhibit rejection, or inhibit arteriosclerosis.“Transplant recipient” refers to the mammalian subject or patient thatreceives the transplanted cells, tissue, or organ, from a donor.Preferred mammalian donors and recipients are human donors andrecipients. The method also may be practiced with pets (dogs, cats),racing animals (dogs, horses); agriculturally important animals (cows,pigs); non-human primates (chimps, gorillas, etc.); and important labanimals (e.g., rodents). The method may be practiced with xenografts offrom one donor species to a different recipient species.

In a related embodiment, the invention is a method for inducingtolerance or inhibiting rejection of a cell, tissue, or organtransplant, or for inhibiting arteriosclerosis in a transplantrecipient, comprising: administering to a mammalian transplant recipienta composition that comprises an nucleic acid that comprises a nucleotidesequence that encodes a growth factor inhibitor, such as an endothelialgrowth factor inhibitor, wherein the nucleic acid is expressible incells of the recipient or expressible in the transplanted cell, tissue,or organ to produce an amount of the endothelial growth factor inhibitoreffective to induce tolerance for the transplant by the recipient, orinhibit rejection, or inhibit arteriosclerosis. To facilitate expressionof the encoded inhibitor, the nucleic acid preferably comprises at leastone expression control sequence operatively connected to the sequencethat encodes the endothelial growth factor inhibitor. Exemplaryexpression control sequences include promoters and enhancers, forexample. In some preferred variations, the method comprisesadministering an expression vector that comprises the nucleic acid tothe transplant recipient. For example, the vector comprises areplication deficient viral vector; such as a retrovirus, an adenovirus,an adeno-associated virus, a vaccinia virus or a herpesvirus vector. Insome variations, the vector is inducible by administration of anexogenous pharmaceutical agent. In other variations, expression of thevector is induced by an endogenous stress in the organ transplantrecipient, such as an elevation of a biological marker correlated withrejection. In still other variations, the vector is constitutivelyexpressed.

In still related embodiments, the method is directed to a method forreducing a transplant recipients dependence or need forimmunosuppressive drugs, by administering such inhibitors to therecipient, in an amount effective to reduce the dose or doing of one ormore immunosuppressant drugs administered to the transplant recipient.

For all embodiments of the invention, whether the therapeutic agent is apolypeptide, an antibody, a polynucleotide, a small molecule, or somecombination thereof, the administered composition preferably furtherincludes a pharmaceutically acceptable carrier. The composition mayinclude one inhibitor or may include a combination of inhibitors, or mayinclude one inhibitor that targets multiple growth factor or growthfactor receptor targets described herein.

Methods of the invention can be practiced with respect to all variationsof transplanted cells or tissue. For example, in some variations, thetransplant is a xenograft, and the method induces tolerance for thexenograft or inhibits xenograft rejection, or reduces xenograft-relatedarteriosclerosis. In other preferred variations, the transplant is anallograft transplant, and the composition is administered in an amounteffective to induce tolerance for the allograft or inhibit alloimmunity,or reduce graft related arteriosclerosis.

In some variations, the transplant may be limited to specified celltypes or to a tissue transplant. For example, the cell or tissuecomprises embryonic stem cells, pluripotent stem cells, hematopoieticprecursor cells, neuronal precursor cells, or endothelial precursorcells. In some variations, the cell or tissue comprises a memberselected from the group consisting of pancreatic islet cells, cardiacmyocytes, bone marrow cells, endothelial cells, and skin cells.

In some variations of the invention, treatment of corneal transplantpatients is specifically excluded from the invention.

In many preferred embodiments, the transplant is an organ or organfragment capable of performing functions of the organ or capable ofregenerating into the organ. For example, the method is practiced on arecipient of at least one transplanted organ, or fragment thereof,selected from the group consisting of a heart, a kidney, a lung, aliver, an intestine, a pancreas, skin, and bone. In some highlypreferred variations, the method is practiced on the recipient of atleast one transplanted organ selected from the group consisting ofheart, lung, liver, and kidney. Treatment of cardiac (heart) transplantrecipients is highly preferred.

All variety of formulations and routes of administration arecontemplated. For example, in some variations, the composition isadministered locally to the transplanted cell, tissue, or organ in therecipient. In some variations, the composition is administeredsystemically to the recipient. In some variations of the method, thecomposition is administered intravenously, intramuscularly, orintraperitoneally, or perorally.

To provide just a few exemplary variations, pharmacological agents maypreferably be administered systemically. Monoclonal antibodies may beadministered intravenously, intramuscularly, or intraperitoneally.Receptor tyrosine kinase inhibitors may be administered perorally orintravenously. Nucleic acid or vectors preferably would be administeredintravascularly or intraparenchymally to the transplanted organ,optionally during the organ procurement.

All variations of timing of administration also are contemplated. Forexample, in some variations, the method further comprises administeringthe composition to the organ or the organ donor before the transplant.(In other variations, the inhibitor composition administered at thisstage contains a different inhibitor from the composition administeredto the recipient.) As described below in detail, donor cells areimplicated in graft rejection, and administering the inhibitors to thedonor organ or donor prior to the transplant is contemplated to havebeneficial effects during the critical perioperative period.

In some variations, the method further comprises repeated administrationof the composition to the recipient.

The composition may be administered to the recipient perioperatively,relative to the transplant operation. The composition may beadministered for varying lengths of time after the transplant operationfor prophylaxis, e.g., 1, 2, 3, 4, 5, 6, 7, 10, 14, 21, 28, 30, 31, 45,56, 60, 90, 120, 180 days post-transplant. All durations from one day tofifty years post-transplant are specifically contemplated. In othervariations, the method is practiced at discrete times to ameliorateacute rejection events. For example, in some variations, the method ispracticed upon detection of symptoms of rejection, and the inhibitor isadministered in an amount effective to alleviate the symptoms. In somevariations, the method comprises a step of screening the organtransplant recipient for symptoms of an acute rejection reaction; wherethe composition that contains the inhibitor is administered to therecipient upon detection of symptoms of acute rejection, in an amounteffective to inhibit the rejection.

A wide variety of growth factor (including endothelial growth factor)inhibitors are described below in detail for practice of the invention.Some of the inhibitors bind to a growth factor or to a receptor, and maybe described below as binding constructs. Other inhibitors may actindirectly, e.g., at the level of effecting gene or protein expression,or inhibiting downstream signaling by an activated receptor.

In some variations, the endothelial growth factor inhibitor comprises acompound that inhibits stimulation of at least one receptor selectedfrom the group consisting of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, andPDGFR-beta by a growth factor ligand of said at least one receptor. Insome highly preferred variations, the endothelial growth factorinhibitor comprises a compound that inhibits stimulation of VEGFR-3 byVEGF-C or inhibits stimulation of VEGFR-3 by VEGF-D.

For example, in some embodiments, the compound comprises an antibodysubstance selected from the group consisting of antibody substances thatimmunoreact with VEGFR-3, antibody substances that immunoreact withVEGF-C, and antibody substances that immunoreact with VEGF-D. The termantibody substance is intended to refer to traditional antibodies andalso to the wide variety of engineered antibody fragments and variantsthat are engineered for therapeutic purposes. For example, exemplarypreferred antibody substances include a humanized antibody, a humanantibody, a monoclonal antibody, a fragment of an antibody that retainsantigen binding characteristics, and a polypeptide that comprises anantigen binding fragment of an antibody. A preferred antibody substanceis a monoclonal antibody (preferably humanized or fully human) thatbinds VEGFR-3 or VEGF-C or VEGF-D and inhibits binding between VEGFR-3and VEGF-C or -D.

Yet another preferred class of inhibitor substances are soluble receptorconstructs that are capable of binding circulating endothelial cellgrowth factor molecules and preventing them from binding and stimulatingreceptors expressed on endothelial or other cell surfaces. Thus, in someembodiments, the endothelial growth factor inhibitor comprises a solublereceptor that binds to at least one endothelial cell growth factor. In apreferred variation, the endothelial growth factor inhibitor comprises asoluble VEGFR-3 polypeptide that binds to VEGF-C or VEGF-D. For example,the soluble VEGFR-3 polypeptide comprises the VEGFR-3 extracellulardomain, or a fragment thereof sufficient to bind VEGF-C or VEGF-D.Exemplary fragments include the first and second immunoglobulin-likedomains of the VEGFR-3; or include the first, second, and thirdimmunoglobulin-like domains of the VEGFR-3. In some preferredvariations, the soluble receptor is fused to an immunoglobulin constantdomain to increase serum half life. Such constructs can be expressedrecombinantly, as fusion proteins.

In still another variation, the inhibitor comprises an antisense nucleicacid or an interfering RNA nucleic acid that inhibits expression of anendothelial cell growth factor or endothelial cell growth factorreceptor. Preferred examples include a short interfering RNA thatinhibits expression of a protein selected from the group consisting ofVEGFR-3, VEGF-C, and VEGF-D; and an antisense nucleic acid that inhibitsexpression of a protein selected from the group consisting of VEGFR-3,VEGF-C, and VEGF-D.

In still other variations of the invention, the inhibitor compoundcomprises bevacizumab (Avastin®) or Ranibizumab (Lucentis®), bothmarketed by Genentech.

In some variations of the invention, a composition that comprises twodifferent inhibitors is administered; or a two or more inhibitorcompositions are administered. Combinations that include an inhibitor ofVEGFR-3 (or VEGF-C or VEGF-D) in combination with an inhibitor of one ormore of the following growth factor receptors (or their ligands) areparticularly preferred: VEGFR-1, VEGFR-2, PDGFR-alpha, and PDGFR-beta.

In still other variations of the invention, the compound is amultivalent inhibitor of two or more receptors selected from the groupconsisting of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta.For example, in some variations, the method of the invention comprisesadministering to the transplant recipient a composition that inhibitsligand binding to VEGFR-2 and inhibits ligand binding to VEGFR-3.

It is contemplated that the inhibitors of the invention protect thetransplant recipient by different mechanisms than traditional orexisting immunosuppressive regimens. In some variations of theinvention, the inhibitors of the invention are co-administered withimmunosuppressive therapy. The combination is expected to provide atleast additive, and preferably synergistic, effects compared to eithertype of agent alone. The synergistic effects can be in the form ofincreased efficacy for survival of the transplant; and also for reducedside effects, possibly due to the need for reduced dosing of theimmunosuppressive agents.

Thus, in some variations, the method of the invention further comprisesadministering an immunosuppressive agent to the organ transplantrecipient. Exemplary classes of immunosuppressive agents includecorticosteriods, calcineurine inhibitors, antiproliferative agents,monoclonal antilymphocyte antibodies, and polyclonal antilymphocyteantibodies. Exemplary immunosuppressive agents include Tacrolimus,Mycophenolic acid, Prednisone, Ciclosporin, Azathioprine, Basiliximab,Cyclosporine, Daclizumab, Muromonab-CD3, Mycophenolate Mofetil,Sirolimus, Methylprednisolone, Atgam, Thymoglobulin, OKT3, Rapamycin,Azathioprine, Cyclosporine, and Interleukin-2 Receptor Antagonist. Theseagents can be administered singly or in combination.

In yet another variation, the method of the invention further comprisingadministering an antibiotic or antifungal agent to the recipient, toprotect the recipient from infections.

In still further variations of the invention, the transplant recipientis helped by pre-treating the donor (or the tissue or organ to betransplanted), with the inhibitory agent. Thus, in some embodiments, themethod of the invention further comprises administering to a donororganism a composition that comprises an endothelial growth factorinhibitor, prior to harvesting a cell, tissue, or organ fortransplantation into the recipient. In other embodiments, the methodfurther comprises contacting a cell, tissue, or organ with a compositionthat comprises an endothelial growth factor inhibitor, prior totransplanting the cell, tissue, or organ into the mammalian organtransplant recipient. In still other embodiments, the method furthercomprises administering to a donor organism, prior to harvesting cells,tissue, or an organ for transplantation, a composition that comprises annucleic acid that comprises a nucleotide sequence that encodes anendothelial growth factor inhibitor, wherein the nucleic acid isexpressible in cells of the tissue or organ to be transplanted. In stillfurther variations, the method further comprises contacting a cell,tissue, or organ with a composition that comprises an nucleic acid thatcomprises a nucleotide sequence that encodes an endothelial growthfactor inhibitor, prior to transplanting the cell, tissue, or organ intothe recipient.

Other embodiments of the invention do not require administration of agrowth factor inhibitor to the recipient at all. For example, oneembodiment of the invention is a method of preparing a donor cell,tissue, or organ for allograft or xenograft transplantation comprisingcontacting the cell, tissue, or organ with a composition that comprisesa growth factor inhibitor, such as an endothelial growth factorinhibitor, prior to transplanting the cell, tissue, or organ into amammalian organ transplant recipient. A related embodiment is a methodof preparing a donor cell, tissue, or organ for allograft or xenografttransplantation comprising contacting the cell, tissue, or organ with acomposition that comprises an nucleic acid that comprises a nucleotidesequence that encodes a growth factor inhibitor, such as an endothelialgrowth factor inhibitor, prior to transplanting the cell, tissue, ororgan into a mammalian organ transplant recipient.

Other variations of the invention are directed to material, useful forpracticing methods of the invention. For example, in one embodiment, theinvention is a composition that comprises an endothelial growth factorinhibitor, an immunosuppressant, and a pharmaceutically acceptablecarrier. Preferably, the inhibitor and the immunosuppressant are presentin the composition in synergistically effective amounts.

In a related variation, the invention is a kit or unit dose in which theinhibitor and the immunosuppressant are packaged together, but not inadmixture.

The present invention relates to compositions and methods of use thereoffor the inhibition of graft (e.g., allograft) rejection andgraft-related arteriosclerosis, and inhibition of other effects ofmembers of the PDGF/VEGF family of growth factors: VEGF-A, VEGF-B,VEGF-C, VEGF-D, VEGF-E, PlGF, PDGF-A, PDGF-B, PDGF-C, and PDGF-D, eachof which is able to bind at least one growth factor receptor tyrosinekinase and stimulate phosphorylation of the same. The compositions ofthe invention include binding constructs that bind one or more PDGF/VEGFmolecules. The binding constructs include one or more binding units.Likewise, many of the inhibitors for use in practicing the invention aredescribed herein as binding units or binding constructs.

In some embodiments, the binding unit comprises a polypeptide, e.g., afragment of a growth factor receptor tyrosine kinase extracellulardomain. The invention also provides nucleic acids encoding such bindingconstructs, and uses thereof. Binding units are not limited to receptorfragments, nor are they limited to polypeptides, but rather comprise anyspecies that binds a growth factor or binds a receptor, and therebyinhibits the circulating growth factor from binding or stimulating thereceptor naturally expressed on the surface of cells. Administration ofthe compositions of the invention to patients inhibits growth factorstimulation of VEGF receptors and/or PDGF receptors (e.g., inhibitsphosphorylation of the receptors) and thereby inhibits biologicalresponses mediated through the receptors including, but not limited to,PDGFR- and/or VEGFR-mediated angiogenesis and lymphangiogenesis.

Each member of the growth factor genus described above binds with highaffinity to, and stimulation phosphorylation of, at least one PDGFreceptor or VEGF receptor (or receptor heterodimer) selected fromVEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta. This statementrefers to well known properties of the growth factors toward theircognate receptors, and is not meant as a limiting feature per se of thebinding constructs of the invention. (For example, VEGF-A has been shownto bind to VEGFR-1 and VEGFR-2 and induce tyrosine phosphorylation ofboth receptors and initiate downstream receptor signaling.) However,preferred binding units of the invention do more than simply bind theirtarget growth factors: a preferred binding construct also inhibits thegrowth factor(s) to which it binds from stimulating phosphorylation ofat least one (and preferably all) of the receptor tyrosine kinases towhich the growth factor(s) bind. Stimulation of tyrosine phosphorylationis readily measured using in vitro cell-based assays andanti-phosphotyrosine antibodies. Because phosphorylation of the receptortyrosine kinases is an initial step in a signaling cascade, it is aconvenient indicator of whether the binding construct is capable ofinhibiting growth factor-mediated signal transduction that leads to cellmigration, cell growth, and other responses. A number of other cellbased and in vivo assays can be used to confirm the growth factorneutralizing properties of binding constructs of the invention.

As described herein, binding constructs can be chemically modified(e.g., heterologous peptide fusions, glycosylation, pegylation, etc.) toimpart desired characteristics, while maintaining their specific growthfactor binding properties. An exemplary peptide fusion comprises aimmunoglobulin constant domain fragment. Exemplary desiredcharacteristics imparted by chemical modifications include increasedserum half life, increased solubility in an aqueous medium, and theability to target a specific cell population, e.g., cancer cells.

Binding constructs and units that are “specific” for a particular growthfactor are binding constructs and units that specifically recognize acirculating, active form of the growth factor. Preferably, the bindingconstructs specifically bind other forms of the growth factors as well.By way of example, VEGF-A exists in multiple isoforms, some of whichcirculate and others of which associate with heparin sulfateproteoglycans on cell surfaces. Binding constructs that are specific forVEGF-A bind to at least a circulating isoform, preferably allcirculating isoforms, and more preferably, bind other major isoforms aswell. By way of another example, VEGF-C is translated as aprepro-molecule with extensive amino-terminal and carboxy-terminalpropeptides that are cleaved to yield a “fully processed” form of VEGF-Cthat binds and stimulates VEGFR-2 and VEGFR-3. Binding constructsspecific for VEGF-C bind to at least the fully processed form of VEGF-C,and preferably also bind to partly processed forms and unprocessedforms.

Additional description is used herein when a more specialized meaning isintended. For example, VEGF-B167 is heparin bound whereas VEGF-B186 isfreely secreted. An binding construct of the invention that minimallybinds the circulating isoform is said to be specific for VEGF-B, andsuch a binding construct preferably also binds the heparin bound form. Abinding construct of the invention that is “specific for heparin-boundVEGF-B” or “specific for VEGF-B167” is a binding construct thatdifferentially recognizes the heparin bound isoform, compared to thefreely circulating isoform. A binding construct of the invention that isspecific for VEGF-B186” is a binding construct that differentiallyrecognizes the circulating form, compared to the heparin bound form.Binding constructs specific for each isoform of a growth factor arecontemplated as components of some embodiments of the binding constructsof the invention.

The designations “first” and “second” and “third” in respect to thebinding units of the binding constructs is for ease and clarity indescription only, and is not meant to signify a particular order, e.g.,order in the amino acid sequence of a polypeptide binding construct.

A binding construct comprising two or more binding units may furthercomprise a linker connecting adjacent binding units. The linker may takeon a number of different forms. Preferably, the linker comprises apeptide which allows adjacent binding units to be linked to form asingle polypeptide.

The invention also includes compositions comprising a polypeptide,binding construct, or nucleic acid encoding the same, together with apharmaceutically acceptable carrier. Such compositions may furthercomprise a pharmaceutically acceptable diluent, adjuvant, or carriermedium.

Nucleic acids (polynucleotides) of the invention include nucleic acidsthat constitute binding units, e.g., aptamers, and also nucleic acidsthat encode polypeptide binding units and constructs, which may be usedfor such applications as gene therapy and recombinant in vitroexpression of polypeptide binding constructs. In some embodiments,nucleic acids are purified or isolated. In some embodiments,polynucleotides further comprise a promoter sequence operativelyconnected to a nucleotide sequence encoding a polypeptide, wherein thepromoter sequence promotes transcription of the sequence that encodesthe polypeptide in a host cell. Polynucleotides may also comprise apolyadenylation sequence. Other nucleic acids of the invention (e.g.,antisense nucleic acids, interfering RNA nucleic acids) operate toinhibit transcription or translation of growth factor genes or receptorgenes.

Vectors comprising polynucleotides are also aspects of the invention.Such vectors may comprise an expression control sequence operativelyconnected to the sequence that encodes the polypeptide, and the vectormay be selected from the group consisting of a lentivirus vector, anadeno-associated viral vector, an adenoviral vector, a liposomal vector,and combinations thereof. In some embodiments, the vector comprises areplication-deficient adenovirus, said adenovirus comprising thepolynucleotide operatively connected to a promoter and flanked byadenoviral polynucleotide sequences. Host cells comprising thepolynucleotides, vectors and other nucleic acids, and methods for usingthe same to express and isolate the binding constructs and units arealso aspects of the invention.

For binding units of a binding construct that comprises an aptamer, theaptamer may be generated by preparing a library of nucleic acids;contacting the library of nucleic acids with a growth factor, whereinnucleic acids having greater binding affinity for the growth factor(relative to other library nucleic acids) are selected and amplified toyield a mixture of nucleic acids enriched for nucleic acids withrelatively higher affinity and specificity for binding to the growthfactor. The processes may be repeated, and the selected nucleic acidsmutated and rescreened, whereby a growth factor aptamer is beidentified. Nucleic acids may be screened to select for molecules thatbind to more than growth factor.

In one aspect of the invention, the binding construct comprises apurified polypeptide comprising an amino acid sequence at least 95%identical to a vascular endothelial growth factor receptor 3(VEGFR-3)fragment, wherein the VEGFR-3 fragment comprises an amino acid sequenceconsisting of a portion of SEQ ID NO: 6, wherein the carboxy-terminalresidue of the fragment is selected from the group consisting ofpositions 211 to 247 of SEQ ID NO: 6. The fragment, and the polypeptidecomprising the same, specifically bind to at least one growth factorselected from the group consisting of human vascular endothelial growthfactor-C (VEGF-C), and human vascular endothelial growth factor-D(VEGF-D). In some embodiments the VEGFR-3 fragments has an aminoterminal amino acid selected from the group consisting of positions 1 to47 of SEQ ID NO: 6. In some embodiments, the polypeptide comprises anamino acid sequence selected from the group consisting of SEQ ID NOS: 36and 38. In some embodiments, the fragment has an amino acid sequenceselected from the group consisting of positions 1-226 and 1-229 of SEQID NO: 6. In some embodiments, the polypeptide is part of a bindingconstruct, and the polypeptide is operatively connected with a secondpolypeptide that binds at least one growth factor selected from thegroup consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF,PDGF-A, PDGF-B, PDGF-C, and PDGF-D. In some embodiments, the secondpolypeptide is selected from the group consisting of a polypeptidecomprising a vascular endothelial growth factor receptor extracellulardomain fragment, a platelet derived growth factor receptor extracellulardomain fragment, and a polypeptide comprising an antigen bindingfragment of an antibody that immunoreacts with the at least one of saidgrowth factors. In some embodiments, at least one of the polypeptides isencoded by a polynucleotide comprising a nucleotide sequence selectedfrom the group consisting of SEQ ID NOS: 35 and 37.

In another aspect of the invention, a binding construct comprises apurified polypeptide comprising an amino acid sequence at least 95%identical to a VEGFR-2 fragment, wherein the VEGFR-2 fragment comprisesan amino acid sequence consisting of a portion of SEQ ID NO: 4, whereinthe amino terminal amino acid of the VEGFR-2 fragment is selected fromthe group consisting of positions 106-145 of SEQ ID NO: 4, wherein thecarboxy terminal amino acid of the VEGFR-2 fragment is selected from thegroup consisting of positions 203 to 240 of SEQ ID NO: 4, and whereinthe VEGFR-2 fragment and the polypeptide bind VEGF-C or VEGF-D. In someembodiments, the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 22, 24, and 26. In someembodiments, the fragment consists of an amino acid sequence selectedfrom the group consisting of residues 118-220, 118-226, and 118-232 ofSEQ ID NO: 4. In some embodiments, the polypeptide is part of a bindingconstruct, and the polypeptide is operatively connected with a secondpolypeptide that binds at least one growth factor selected from thegroup consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF,PDGF-A, PDGF-B, PDGF-C, and PDGF-D. In some embodiments, the secondpolypeptide is selected from the group consisting of a polypeptidecomprising a vascular endothelial growth factor receptor extracellulardomain fragment, a platelet derived growth factor receptor extracellulardomain fragment, and a polypeptide comprising an antigen bindingfragment of an antibody that immunoreacts with the at least one of saidgrowth factors. In some embodiments, at least one of the polypeptides isencoded by a polynucleotide comprising a nucleotide sequence selectedfrom the group consisting of SEQ ID NOS: 21, 23, and 25.

In still another aspect, the invention provides a binding constructcomprising a first polypeptide operatively connected to a secondpolypeptide. The first and second polypeptides each binds at least onegrowth factor selected from the group consisting of VEGF-A, VEGF-B,VEGF-C, VEGF-D, VEGF-E, PlGF, PDGF-A, PDGF-B, PDGF-C, and PDGF-Dpolypeptides. The amino acid sequence of the first polypeptide differsfrom the amino acid sequence of the second polypeptide. The first andsecond polypeptides comprise members independently selected from thegroup consisting of:

(a) a polypeptide comprising an amino acid sequence at least 90%identical to the VEGFR-1 extracellular domain amino acid sequencecomprising positions 27-758 of SEQ ID NO: 2;

(b) a fragment of (a) that binds VEGF-A, VEGF-B, or PlGF;

(c) a polypeptide comprising an amino acid sequence at least 90%identical to the VEGFR-2 extracellular domain amino acid sequencecomprising positions 20-764 of SEQ ID NO: 4;

(d) a fragment of (c) that binds VEGF-A, VEGF-C, VEGF-E or VEGF-D;

(e) a polypeptide comprising an amino acid sequence at least 90%identical to the VEGFR-3 extracellular domain amino acid sequencecomprising residues 24-775 of SEQ ID NO: 6;

(f) a fragment of (e) that binds VEGF-C or VEGF-D;

(g) a polypeptide comprising an amino acid sequence at least 90%identical to the neuropilin-1 extracellular domain amino acid sequencecomprising residues 22-856 of SEQ ID NO: 113;

(h) a fragment of (g) that binds VEGF-A, VEGF-B, VEGF-C, VEGF-E, orPlGF;

(i) a polypeptide comprising an amino acid sequence at least 90%identical to the neuropilin-2 extracellular domain amino acid sequencecomprising residues 21-864 of SEQ ID NO: 115;

(j) a fragment of (i) that binds VEGF-A, VEGF-C, or PlGF;

(k) a polypeptide comprising an amino acid sequence at least 90%identical to the platelet derived growth factor receptor alphaextracellular domain amino acid sequence comprising residues 24-524 ofSEQ ID NO: 117;

(l) a fragment of (k) that binds PDGF-A, PDGF-B, or PDGF-C;

(m) a polypeptide comprising an amino acid sequence at least 90%identical to the platelet derived growth factor beta extracellulardomain amino acid sequence comprising residues 33 to 531 of SEQ ID NO:119;

(n) a fragment of (m) that binds PDGF-B or PDGF-D; and

(o) a polypeptide comprising an antigen binding fragment of an antibodythat binds to at least one growth factor selected from the groupconsisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF, PDGF-A,PDGF-B, PDGF-C, and PDGF-D.

Still further examples of polypeptides that comprise binding units ofthe invention are antibodies and antibody fragments that immunoreactwith one or more receptors selected form VEGFR-1, VEGFR-2, VEGFR-3,PDGFR-alpha, and PDGFR-beta.

In one embodiment, the binding construct of the invention comprises afirst polypeptide comprising a fragment of a polypeptide comprising anamino acid sequence at least 90% identical to the VEGFR-2 extracellulardomain amino acid sequence comprising positions 20-764 of SEQ ID NO: 4,wherein the fragment binds VEGF-A, VEGF-C, VEGF-E or VEGF-D. Optionally,the binding construct further comprises a second polypeptide comprisinga fragment of a polypeptide comprising an amino acid sequence at least90% identical to the VEGFR-1 extracellular domain amino acid sequencecomprising positions 27-758 of SEQ ID NO: 2; wherein the fragment bindsVEGF-A, VEGF-B, or PlGF. Additionally, the binding construct optionallyfurther comprises a third polypeptide operatively connected to the firstor second polypeptide, wherein the third polypeptide comprises afragment of a polypeptide comprising an amino acid sequence at least 90%identical to the VEGFR-3 extracellular domain amino acid sequencecomprising residues 24-775 of SEQ ID NO: 6, wherein the fragment bindsVEGF-C or VEGF-D.

As described herein in greater detail, the extracellular domain of VEGFRor PDGFR have immunoglobulin-like domain structure. In a relatedembodiment, the binding construct of the invention comprises a first,second and third polypeptide as described above, wherein: (a) the firstpolypeptide comprises an amino acid sequence at least 90% identical to afragment of the VEGFR-2 extracellular domain, wherein the fragmentcomprises immunoglobulin-like domain 2 amino acid sequence; (b) thesecond polypeptide comprises an amino acid sequence at least 90%identical to a fragment of the VEGFR-1 extracellular domain, wherein thefragment comprises immunoglobulin-like domain 3 amino acid sequence; and(c) the third polypeptide comprises an amino acid sequence at least 90%identical to a fragment of the VEGFR-3 extracellular domain, whereinsaid fragment comprises VEGFR-3 immunoglobulin-like domain 1 amino acidsequence.

In another aspect, the invention involves use of a binding constructcomprising: a) a first amino acid sequence at least 90% identical to afragment of the VEGFR-3 extracellular domain, wherein said fragmentcomprises VEGFR-3 immunoglobulin-like domain 1 amino acid sequence; (b)a second amino acid sequence at least 90% identical to a fragment of theVEGFR-2 extracellular domain, wherein the fragment comprisesimmunoglobulin-like domain 2 amino acid sequence; and, (c) a third aminoacid sequence at least 90% identical to a fragment of the VEGFR-1extracellular domain, wherein the fragment comprises immunoglobulin-likedomain 3 amino acid sequence; wherein the first, second, and third aminoacid sequences are operatively connected, and wherein the bindingconstruct binds to at least VEGF-A and VEGF-C. In one embodiment, thebinding construct comprises an amino acid sequence at least 95%identical to the amino acid sequence set out in SEQ ID NO: 128. In arelated embodiment, the binding construct comprises the amino acidsequence of SEQ ID NO: 128.

In some embodiments, the binding construct of the invention comprises afirst polypeptide comprising a fragment of a polypeptide comprising anamino acid sequence at least 90% identical to the VEGFR-3 extracellulardomain amino acid sequence comprising residues 24-775 of SEQ ID NO: 6,wherein the fragment binds VEGF-C or VEGF-D. It is contemplated that thebinding construct of the invention comprises a second polypeptidecomprising a fragment of a polypeptide comprising an amino acid sequenceat least 90% identical to the VEGFR-2 extracellular domain amino acidsequence comprising positions 20-764 of SEQ ID NO: 4, wherein thefragment binds VEGF-A, VEGF-C, VEGF-E or VEGF-D.

In a related embodiment, the binding construct of the inventioncomprises a first and second polypeptide as described above, wherein:(a) the first polypeptide comprises an amino acid sequence at least 90%identical to a fragment of the VEGFR-3 extracellular domain, whereinsaid fragment comprises VEGFR-3 immunoglobulin-like domain 1 amino acidsequence; and, (b) the second polypeptide comprises an amino acidsequence at least 90% identical to a fragment of the VEGFR-2extracellular domain, wherein the fragment comprises immunoglobulin-likedomains 2 and 3 amino acid sequence.

In another aspect, the invention provides a binding constructcomprising: a) a first amino acid sequence at least 90% identical to afragment of the VEGFR-3 extracellular domain, wherein said fragmentcomprises VEGFR-3 immunoglobulin-like domain 1 amino acid sequence; and,(b) a second amino acid sequence at least 90% identical to a fragment ofthe VEGFR-2 extracellular domain, wherein the fragment comprisesimmunoglobulin-like domain 2 amino acid sequence; and animmunoglobulin-like domain 3 amino acid sequence; wherein the first,second, and third amino acid sequences are operatively connected, andwherein the binding construct binds to at least VEGF-A and VEGF-C. It isfurther contemplated that the construct binds VEGF-D. In one embodiment,the binding construct comprises an amino acid sequence at least 95%identical to the amino acid sequence set out in SEQ ID NO: 125. In arelated embodiment, the binding construct comprises the amino acidsequence of SEQ ID NO: 125.

In some variations, the binding unit or units of a binding compriseantibodies or antibody antigen binding fragments. In some embodiments,the binding construct comprises at least one non-antigen bindingfragment binding unit. In some embodiments, the binding units allcomprise antigen binding fragments of antibodies. Exemplary Bispecificantibodies are provided in U.S. patent application Ser. No. 11/075,400,published as U.S. Patent Publication No. 2005/0282233, and related,co-filed International Patent Application No. PCT/US2005/007742,published as WO 2005/087812 (Attorney Docket No. 28967/39820B), bothapplications incorporated herein by reference it their entirety.Antibodies that target the growth factors identified herein, andantibodies that target the receptors indentified herein, all are usefulfor practicing the invention. Monoclonal antibody therapeutics arepreferred. Humanized and fully human antibodies are highly preferred, asare fragments of such antibodies.

One aspect of the invention is a method for inhibiting allograftrejection or graft-related arteriosclerosis comprising administering toa mammalian subject in need of said inhibition a binding constructaccording to the invention, in an amount effective to inhibit theallograft rejection or the arteriosclerosis.

The method may also comprise the step of screening an organ transplantrecipient mammal to identify elevated levels of at least one growthfactor selected from the group consisting of VEGF-A, VEGF-B, VEGF-C,VEGF-D, VEGF-E, PlGF, PDGF-A, PDGF-B, PDGF-C, and PDGF-D polypeptides.In some embodiments, the screening step comprises obtaining a serumsample, a fluid sample, or a tissue sample from the transplanted organand detecting elevated levels of at least one growth factor selectedfrom the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E,PlGF, PDGF-A, PDGF-B, PDGF-C, and PDGF-D polypeptides, or elevatedlevels of at least one receptor capable of binding the same.

The methods of the invention may also be carried out with anothertherapeutic. For example, other therapeutics that may be used alone, orin combination with the binding constructs of the invention, includeanti-sense RNA, RNA interference, bispecific antibodies, other antibodytypes, and small molecules, e.g., chemotherapeutic agents, which targetgrowth factors and/or their receptors. Combination therapies arepreferably synergistic, but they need not be, and additive therapies arealso considered aspects of the invention.

In addition to their use in methods, the binding constructs may becombined or packaged with other therapeutics in kits or as unit doses.

This summary of the invention is not intended to be limiting orcomprehensive, and additional embodiments are described in the drawingsand detailed description, including the examples. All such embodimentsare aspects of the invention. Moreover, for the sake of brevity, variousdetails that are applicable to multiple embodiments have not beenrepeated for every embodiment. Variations reflecting combinations andrearrangements of the embodiments described herein are intended asaspects of the invention. In addition to the foregoing, the inventionincludes, as an additional aspect, all embodiments of the inventionnarrower in scope in any way than the variations specifically mentionedabove. For example, for aspects described as a genus or range, everysubgenus, subrange or species is specifically contemplated as anembodiment of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that VEGFR-3 inhibition markedly improves long-termsurvival of rat cardiac allografts in suboptimally-immunosuppressedrecipients.

DETAILED DESCRIPTION

The present invention provides binding constructs, compositions, andmaterials and methods for making and using the same. The bindingconstructs bind growth factors that have been shown or are hypothesizedto contribute to allograft rejection or arteriosclerosis in allograftreceipients in vivo, and are useful for inhibiting those effects.

I. BINDING CONSTRUCTS

For the purposes of this invention, a “binding construct” comprises oneor more binding units associated with each other by covalent or otherforms of attachment. A “binding unit” binds a growth factor receptor ora growth factor ligand, i.e., binds to one or more growth factorpolypeptides or growth factor receptor polypeptdies, and preferably doesso with high affinity. A binding unit preferably comprises at least onepeptide or polypeptide, but other embodiments are possible as well,including organic small molecules, aptamers, and combinations of thesame. While a binding unit preferably comprises a single polypeptide, itmay comprise multiple polypeptides if a single polypeptide is notsufficient for binding a particular growth factor. When more than onebinding unit or polypeptide segment is in a given binding construct, thebinding units may be joined directly (i.e., through a covalent bond,e.g., a peptide, ester, or sulfhydrl bond, or non-covalently, e.g.,hydrophobically) together via a linker. A binding construct may furtherinclude a heterologous peptide or other chemical moieties. Suchadditions are can modify binding construct properties such as stability,solubility, toxicity, serum half-life, immunogenicity, detectability, orother properties.

The term “high affinity” is used in a physiological context pertainingto the relative affinity of the binding construct for the growth factorligand(s) or receptor(s) in vivo in a mammal, such as a laboratory testanimal, a domesticated farm or pet animal, or a human. The targetedgrowth factors of the invention, e.g., the VEGF/PDGF family members,have characteristic affinities for their receptors in vivo, typicallymeasured in terms of sub-nanomolar dissociation constants (K_(d)). Forthe purposes of this invention, a binding construct can bind to itstarget growth factor(s) or receptor(s) with a K_(d) less than or equalto 1000 times the K_(d) of the natural growth factor-receptor pair,while retaining the specificity of the natural pair. A binding unit thatbinds a growth factor with a K_(d) less than or equal to 10 times theK_(d) of the natural growth factor-receptor pair, while retaining thespecificity of the natural pair, is considered high affinity. While highaffinity is preferred, it is not a requirement. In a preferredembodiment, the affinity of the binding unit for the growth factor orreceptor equals or exceeds the affinity of the natural receptor for thegrowth factor (or vice versa). Such affinities may be readily determinedusing conventional techniques, such as by using a BIAcore instrument orby radioimmunoassay using radiolabeled target antigen. Affinity data maybe analyzed, for example, by the method of Scatchard et al., Ann N.Y.Acad. Sci., 51:660 (1949).

By binding activity is meant the ability to bind to a ligand, receptor,or binding construct, and does not require the retention of biologicalactivity in so far as enzymatic activity or signaling is concerned.Binding may include either binding to a monomer or a dimer, homodimersor heterodimers, whether of receptors or ligands. Polypeptides for useaccording to the present invention can be used in the form of a proteindimer, particularly a disulfide-linked dimer. Mechanistic descriptionsof binding constructs, e.g., as ligand traps, are not meant to belimiting. For example, a binding construct comprising a receptorextracellular domain fragment may function by forming inactive dimerswith an endogenous receptor monomer.

In some embodiments, a binding construct comprises a first binding unit(e.g., a polypeptide) operatively associated with a second binding unit(e.g., a polypeptide), wherein each binding unit binds a growth factorselected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGF-E, PlGF, PDGF-A, PDGF-B, PDGF-C, PDGF-D, D1701 VEGF, NZ2 VEGF, NZ7VEGF, and fallotein. In some embodiments the first and second bindingunits act together to bind a single ligand molecule (wherein the ligandmay comprise a monomer or dimer). In some embodiments, the binding unitsact independently, i.e., each polypeptide binds a separate ligandmolecule. In some embodiments, the first and second binding units arecapable of either acting together or acting independently to bind one ormore ligand polypeptides. In some embodiments, a binding unit of a firstbinding construct is able to interact with a binding unit on a secondbinding construct, e.g., to form dimers between binding units.

In some embodiments, a binding construct comprises a first binding unitoperatively associated with a second binding unit, wherein each bindingunit binds to a growth factor receptor selected from the groupconsisting of VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, PDGFR-beta, andthe neuropilins.

In some embodiments, the binding construct comprises a first polypeptideoperatively connected to a second polypeptide, wherein the first andsecond polypeptides each binds at least one growth factor selected fromthe group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and PlGFpolypeptides; or bind at least one growth factor receptor selected fromVEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, PDGFR-beta, and the neuropilins,wherein the amino acid sequence of the first polypeptide differs fromthe amino acid sequence of the second polypeptide; and wherein the firstand second polypeptides comprise members independently selected from thegroup consisting of:

(a) a polypeptide comprising an amino acid sequence at least 35%identical to the VEGFR-1 extracellular domain amino acid sequencecomprising positions 27-758 of SEQ ID NO: 2;

(b) a fragment of (a) that binds VEGF-A, VEGF-B, or PlGF;

(c) a polypeptide comprising an amino acid sequence at least 35%identical to the VEGFR-2 extracellular domain amino acid sequencecomprising positions 20-764 of SEQ ID NO: 4;

(d) a fragment of (c) that binds VEGF-A, VEGF-C, VEGF-E or VEGF-D;

(e) a polypeptide comprising an amino acid sequence at least 35%identical to the VEGFR-3 extracellular domain amino acid sequencecomprising residues 24-775 of SEQ ID NO: 6;

(f) a fragment of (e) that binds VEGF-C or VEGF-D;

(g) a polypeptide comprising an amino acid sequence at least 35%identical to the neuropilin-1 extracellular domain amino acid sequencecomprising residues 22-856 of SEQ ID NO: 113;

(h) a fragment of (g) that binds VEGF-A, VEGF-B, VEGF-C, VEGF-E, orPlGF;

(i) a polypeptide comprising an amino acid sequence at least 35%identical to the neuropilin-2 extracellular domain amino acid sequencecomprising residues 21-864 of SEQ ID NO: 115;

(j) a fragment of (i) that binds VEGF-A, VEGF-C, or PlGF;

(k) a polypeptide comprising an amino acid sequence at least 35%identical to the platelet derived growth factor receptor alphaextracellular domain amino acid sequence comprising residues 24-524 ofSEQ ID NO: 117;

(l) a fragment of (k) that binds PDGF-A, PDGF-B, or PDGF-C;

(m) a polypeptide comprising an amino acid sequence at least 35%identical to the platelet derived growth factor beta extracellulardomain amino acid sequence comprising residues 33 to 531 of SEQ ID NO:119;

(n) a fragment of (m) that binds PDGF-B or PDGF-D;

(o) an antibody that binds to at least one growth factor or receptorselected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D,VEGF-E, PlGF, PDGF-A, PDGF-B, PDGF-C, PDGF-D; VEGFR-1, VEGFR-2, VEGFR-3,PDGFR-alpha, and PDGFR-beta;

(p) a polypeptide comprising an antigen binding fragment of an antibodythat binds to at least one growth factor selected from the groupconsisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF, PDGF-A,PDGF-B, PDGF-C, and PDGF-D; or of an antibody that binds to at least onegrowth factor receptor selected from the group consisting of VEGFR-1,VEGFR-2, VEGFR-3, PDGFR-alpha, and PDGFR-beta;

(q) a polypeptide that binds at least one growth factor selected fromthe group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF,PDGF-A, PDGF-B, PDGF-C, and PDGF-D polypeptides, wherein the polypeptideis generated using phage display;

(r) compounds that comprises peptide fragments of one or more of VEGF-A,VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlGF, PDGF-A, PDGF-B, PDGF-C, andPDGF-D, and that inhibit the binding between such growth factors andtheir receptors; and

(s) an organic molecule that mimics the binding properties of (a)-(r).

In some embodiments, the binding units all comprise antigen bindingfragments. Exemplary bispecific antibodies are provided in U.S. patentapplication Ser. No. 11/075,400, published as U.S. Patent PublicationNo. 2005/0282233, and related International Patent Application No.PCT/US2005/007742, published as WO 2005/087812 (Attorney Docket No.28967/39820B), both applications incorporated herein by reference ittheir entirety.

In some embodiments, one or more of the polypeptides of a bindingconstruct is replaced with another type of molecule, e.g., a nucleicacid, that mimics the binding properties of any of the polypeptidesdescribed above in (a) through (p). Such nucleic acids include, forexample, aptamers.

A. Binding Units

The growth factors that are the targets of the binding constructs of theinvention exert their physiological effects in vivo by binding to theextracellular domains of growth factor receptors. Accordingly, growthfactor receptors and fragments thereof constitute examples of bindingunits. Exemplary human nucleotide and amino acid sequences, for relevantligands and receptors are set forth in the sequence listing assummarized below:

TABLE 1A RECEPTOR SEQUENCES RECEPTOR SEQ ID NOS: VEGFR-1 1 and 2 VEGFR-23 and 4 VEGFR-3 short 5 and 6 VEGFR-3 long 120 and 121 PDGFR-α 116 and117 PDGFR-β 118 and 119 Neuropilin-1 112 and 113 Neuropilin-2 114 and115

TABLE 1B LIGAND SEQUENCES LIGAND SEQ ID NOS: VEGF-A 80 and 81 VEGF-A 232isoform 90 and 91 VEGF-B isoform 1 94 and 95 VEGF-B isoform 2 96 and 97VEGF-C 82 and 83 VEGF-D 86 and 87 VEGF-E (NZ7) 88 and 89 P1GF 84 and 85D1701 VEGF 92 and 93 PDGF-A 98 and 99 PDGF-B 100 and 101 PDGF-C 102 and103 PDGF-D 104 and 105

Other VEGF growth factors members include snake venom VEGFs (e.g., EMBL.AY033151, AY033152, and AY42981), various VEGF-E (orf virus VEGFhomologs, some of which are presented in Table 1B) molecules includingVEGF-E NZ2 [S67520], VEGF-E NZ7, VEGF-E D1701, VEGF-E Orf-11, and VEGF-EOV-IA82. [See generally, WO 00/25085.]

Members of the PDGF/VEGF family are characterized by a number ofstructural motifs including a conserved PDGF motif defined by thesequence: P—[PS]-C-V—X(3)-R—C—[GSTA]-G-C—C (SEQ ID NO: 111), where thebrackets indicate a variable position that can be any one of the aminoacids within the brackets. The number contained within the parenthesesindicates the number of amino acids that separate the “V” and “R”residues. This conserved motif falls within a large domain of 70-150amino acids defined in part by eight highly conserved cysteine residuesthat form inter- and intramolecular disulfide bonds. This domain forms acysteine knot motif composed of two disulfide bonds which form acovalently linked ring structure between two adjacent B strands, and athird disulfide bond that penetrates the ring [see for example, FIG. 1in Muller et al., Structure 5:1325-1338 (1997)], similar to that foundin other cysteine knot growth factors, e.g., transforming growthfactor-β (TGF-β). The amino acid sequence of all known PDGF/VEGFproteins, with the exception of VEGF-E, contains the PDGF domain. ThePDGF/VEGF family proteins are predominantly secreted glycoproteins thatform either disulfide-linked or non-covalently bound homo- orheterodimers whose subunits are arranged in an anti-parallel manner[Stacker and Achen, Growth Factors 17:1-11 (1999); Muller et al.,Structure 5:1325-1338 (1997)]. Binding constructs of the inventioninclude those that bind VEGF/PDGF growth factor monomers, homodimers,and heterodimers.

The VEGF subfamily is composed of members that share a VEGF homologydomain (VHD) characterized by the sequence:C—X(22-24)-P—[PSR]-C-V—X(3)—R—C—[GSTA]-G-C—C—X(6)-C—X(32-41)-C. (SEQ ID:110) The VHD domain, determined through analysis of the VEGF subfamilymembers, comprises the PDGF motif but is more specific. The VEGFsubfamily of growth factors and receptors regulate the development andgrowth of the vascular endothelial system. VEGF family members include,but are not limited to VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF [Li, X.and U. Eriksson, “Novel VEGF Family Members: VEGF-B, VEGF-C and VEGF-D,”Int. J. Biochem. Cell. Biol., 33(4):421-6 (2001))] Other VEGFs arebacterial or viral, the “VEGF-Es.” Other VEGFs are derived from snakevenom, the “NZ” series. [See e.g., Komori, et al. Biochemistry,38(36):11796-803 (1999); Gasmi, et al., Biochem Biophys Res Commun,268(1):69-72 (2002); Gasmi, et al., J Biol Chem; 277(33):29992-8 (2002);de Azevedo, et al., J. Biol. Chem., 276: 39836-39842 (2001)].

At least seven cell surface receptors that interact with PDGF/VEGFfamily members have been identified. These include PDGFR-α [See e.g.,GenBank Acc. No. NM006206; Swiss Prot No. P16234], PDGFR-β [See e.g.,GenBank Acc. No. NM002609; Swiss Prot. No. P09619], VEGFR-1/Flt-1(fms-like tyrosine kinase-1; hereinafter “R-1”) [GenBank Acc. No.X51602; De Vries, et al., Science 255:989-991 (1992)]; VEGFR-2/KDR/Flk-1(kinase insert domain containing receptor/fetal liver kinase-1,hereinafter “R-2”) [GenBank Acc. Nos. X59397 (Flk-1) and L04947 (KDR);Terman, et al., Biochem. Biophys. Res. Comm. 187:1579-1586 (1992);Matthews, et al., Proc. Natl. Acad. Sci. USA 88:9026-9030 (1991)];VEGFR-3/Flt4 (fms-like tyrosine kinase 4; hereinafter “R-3”) [U.S. Pat.No. 5,776,755 and GenBank Acc. No. X68203 and S66407; Pajusola et al.,Oncogene 9:3545-3555 (1994); Hughes, et al., J. Mol. Evol. 52(2):77-79(2001); Pajusola, et al., Oncogene 8(11):2931-37) (1993); Borg, et al.,Oncogene 10(5):973-984 (1995), neuropilin-1 [Gen Bank Acc. No.NM003873], and neuropilin-2 [Gen Bank Acc. No. NM003872; SwissProt060462]. The two PDGF receptors mediate signaling of PDGFs. Non-humanVEGF and PDGF receptors may also be employed as part of the invention,e.g., chicken VEGFR-1 may be used alone or in hybrid form with human R-1for improved expression.

VEGF121, VEGF165, VEGF-B, PlGF-1 and PlGF-2 bind VEGF-R1; VEGF121,VEGF145, VEGF165, (fully processed mature) VEGF-C, (fully processedmature) VEGF-D, VEGF-E, and NZ2 VEGF bind VEGF-R2; VEGF-C and VEGF-Dbind VEGFR-3; VEGF165, VEGF-C, PlGF-2, and NZ2 VEGF bind neuropilin-1;and VEGF165 and VEGF-C binds neuropilin-2. [Neufeld, et al., FASEB. J.13:9-22 (1999); Stacker and Achen, Growth Factors 17:1-11 (1999);Ortega, et al., Fron. Biosci. 4:141-152 (1999); Zachary, Intl. J.Biochem. Cell. Bio. 30:1169-1174 (1998); Petrova, et al., Exp. Cell.Res. 253:117-130 (1999); U.S. Pat. Appl. Pub. No. 20030113324]. PDGF-A,PDGF-B, and PDGF-C bind PDGFR-α. PDGF-B and PDGF-D bind PDGF-β.

Both the ligands and the receptors generally exist as dimers, includingboth homodimers and heterodimers. Such dimers can influence binding. Forexample, for the PDGFs, PDGF-AA binds PDGFR-α/α. PDGF-AB and PDGF-CCbind PDGFR-α/α and PDGFR-α/β. PDGFR-BB binds both of the homodimers andthe heterodimeric PDGF receptor. PDGF-DD binds PDGF receptorheterodimers and beta receptor homodimers. [See, e.g., Pietras, et al.,Cancer Cell, 3:439-443 (2003).] VEGF-A can heterodimerize with VEGF-Band PlGF. The VEGFs, PDGFs, and PlGFs, may exist as two or moreisoforms, e.g., splice variants, and not all isoforms of a particulargrowth factor will share the same binding profile, or ability todimerize with particular molecules. Certain isoforms of the same growthfactor may also dimerize with each other. For example the 167 and 186isoforms of VEGF-B can heterodimerize with each other.

Growth factor receptor tyrosine kinases generally comprise threeprincipal domains: an extracellular domain, a transmembrane domain, andan intracellular domain. The extracellular domain binds ligands, thetransmembrane domain anchors the receptor to a cell membrane, and theintracellular domain possesses one or more tyrosine kinase enzymaticdomains and interacts with downstream signal transduction molecules. Thevascular endothelial growth factor receptors (VEGFRs) and plateletderived growth factor receptors (PDGFRs) bind their ligand through theirextracellular domains (ECDs), which are comprised of multipleimmunoglobulin-like domains (Ig-domains). Ig-domains are identifiedherein using the designation “D#.” For example “D1” refers to the firstIg-domain of a particular receptor ECD. “D1-3” refers to a constructcontaining at least the first three Ig-domains, and intervening sequencebetween domains 1 and 2 and 2 and 3, of a particular construct. Table 2defines the boundaries of the Ig-domains for VEGFR-1, VEGFR-2, andVEGFR-3 of the invention. These boundaries are significant as theboundaries chosen can be used to form constructs, and so can influencethe binding properties of the resulting constructs. This relationship isdiscussed in Example 1.

The complete ECD of PDGFRs and VEGFRs is not required for ligand (growthfactor) binding. The ECD of VEGFR-1 (R-1) and VEGFR-2 (R-2) consists ofseven Ig-like domains and the ECD of VEGFR-3 (R-3) has six intactIg-like domains—D5 of R-3 is cleaved post-translationally into disulfidelinked subunits leaving VEGFR-3. Veikkola, T., et al., Cancer Res.60:203-212 (2000). In general, receptor fragments of at least the firstthree Ig-domains for this family are sufficient to bind ligand. ThePDGFRs have five Ig-domains.

TABLE 2 IMMUNOGLOBULIN-LIKE DOMAINS FOR VEGFR-1, VEGFR-2 AND VEGFR-3 R-1R-1 R-2 R-2 R-3 R-3 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO:2 NO: 3 NO: 4 NO: 5 NO: 6 positions positions positions positionspositions positions D1 394-580  49-111 145-316  48-105 158-364  47-115D2 709-880 154-211 436-610 145-203 479-649 154-210 D3  990-1192 248-315724-931 241-310 761-961 248-314 D4 1303-1474 352-409 1039-1204 346-4011070-1228 351-403 D5 1957-1864 450-539 1321-1600 440-533 1340-1633441-538 D6 1966-2167 573-640 1699-1936 566-645 1739-1990 574-657 D72281-2452 678-735 2050-2221 683-740 2102-2275 695-752

In some embodiments, a binding unit of a binding construct comprises theECD of a growth factor receptor. A binding unit may comprise at leastone Ig-domain of a VEGFR as described in Table 2, to as many as seven.Ig-domain information for PDGFR-α and PDGFR-β is provided in Lokker, etal., J. Biol. Chem. 272: 33037-33044 (1997), which is incorporated byreference in its entirety. A binding unit may include sequence beforethe N-terminal most Ig-domain, may include sequence beyond theC-terminal most Ig-domain, and may include sequence between theIg-domains as well. Binding units may also comprise variants, e.g., withone or more amino acid substitutions, additions, or deletions of anamino acid residue. Binding units also may comprise chimeras, e.g.,combinations of Ig-domains from different receptors. In someembodiments, the first or second polypeptide comprises a receptorfragment comprising at least the first three Ig domains of a receptortyrosine kinase.

The binding of a binding unit to a particular growth factor ligandrefers to the ability to bind at least one natural isoform of at leastone target growth factor, especially processed forms that are secretedfrom cells and circulate in vivo and/or bind heparin moieties. Forexample, “capable of binding VEGF-A” refers to the ability to bind atleast one isoform of VEGF-A under physiological conditions. At leastfive human VEGF-A isoforms of 121, 145, 165, 189 or 206 amino acids inlength (VEGF121-VEGF206), encoded by distinct mRNA splice variants, havebeen described, all of which are capable of stimulating mitogenesis inendothelial cells. [See generally, Ferrara, J. Mol. Med. 77:527-543(1999).] Two VEGF-β isoforms generated by alternative mRNA splicingexist, VEGF-B186 and VEGF-B167, with the first isoform accounting forabout 80% of the total VEGF-B transcripts [Li, X., et al., GrowthFactor, 19:49-59 (2001); Grimmond, et al., Genome Res., 6:124-131(1996); Olofsson, et al., J. Biol. Chem., 271:19310-19317 (1996).] Threeisoforms of PlGF produced by alternative mRNA splicing have beendescribed [Hauser, et al., Growth Factors 9:259-268 (1993); Maglione, etal., Oncogene 8:925-931 (1993)]. PDGF-A and PDGF-B can homodimerize orheterodimerize to produce three different isoforms: PDGF-AA, PDGF-AB, orPDGF-BB.

The term “identity”, as known in the art, refers to a relationshipbetween the sequences of two or more polypeptide molecules or two ormore nucleic acid molecules, as determined by comparing the sequences.In the art, “identity” also means the degree of sequence relatednessnucleic acid molecules or polypeptides sequences, as the case may be, asdetermined by the match between strings of two or more nucleotide or twoor more amino acid sequences. “Identity” measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by particular a mathematical model ofcomputer program (i.e., “algorithms”). Appropriate algorithms fordetermining the percent identies of the invention include BLASTP andBLASTN, using the most common and accepted default parameters.

1. VEGFR-1-Derived Binding Units

In some embodiments, a binding unit comprises a polypeptide similar oridentical in amino acid sequence to a VEGFR-1 polypeptide or fragmentthereof, preferably from the same species as the targeted growthfactor(s). Thus, for binding to human growth factors, a binding unitpreferably comprises a polypeptide that comprises an amino acid similaror identical to a fragment of SEQ ID NO: 2, wherein the fragment and thepolypeptide binds one or more growth factors selected from the groupconsisting of VEGF-A, VEGF-B, and PlGF. The fragment minimally comprisesenough of the VEGFR-1 sequence to bind the ligand, and may comprise thecomplete receptor. Extracellular domain fragments are preferred.Preferred polypeptides have an amino acid sequence at least 80%identical to a ligand binding fragment thereof. Fragments that are moresimilar, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, or 100% are highly preferred. Fragments that are 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, and 75% are also contemplated.

Preferred polypeptides may also be described as having an amino acidsequence encoded by a nucleic acid sequence at least 80% identical to afragment of SEQ ID NO:1 encoding a ligand binding fragment of VEGFR-1.Nucleic acid fragments that are more similar, e.g., 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% are highly preferred.Fragments that are 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75% arealso contemplated. A genus of similar polypeptides can alternatively bedefined by the ability of encoding polynucleotides to hybridize to thecomplement of a nucleotide sequence that corresponds to the cDNAsequence encoding the R-1 receptor. For example, a preferred bindingunit polypeptide comprises an amino acid sequence that binds one or moreR-1 ligands and that is encoded by a nucleotide sequence that hybridizesto the complement of SEQ ID NO: 1 under moderately or highly stringentconditions discussed herein.

Exemplary R1 fragments for use as binding unit polypeptides (or for useas a starting point for designing R-1 analogs) have an amino terminalresidue selected from the group consisting of positions 1 to 129 of SEQID NO: 2, and a carboxy terminal residue selected from the groupconsisting of positions 229 to 758 of SEQ ID NO: 2, wherein the VEGFR-1fragment binds at least one of VEGF-A, VEGF-B, and PlGF.

2. VEGFR-2-Derived Binding Units

In some embodiments, a binding unit comprises a polypeptide similar oridentical in amino acid sequence to a VEGFR-2 polypeptide or fragmentthereof, preferably from the same species as the targeted growthfactor(s). Thus, for binding to human growth factors, a binding unitpreferably comprises a polypeptide that comprises an amino acid similaror identical to a fragment of SEQ ID NO: 4, wherein the fragment and thepolypeptide binds one or more growth factors selected from the groupconsisting of VEGF-A, VEGF-C, VEGF-D, or VEGF-E. The fragment minimallycomprises enough of the VEGFR-2 sequence to bind the ligand, and maycomprise the complete receptor. Extracellular domain fragments arepreferred. Preferred polypeptides have an amino acid sequence at least80% identical to a ligand binding fragment thereof. Fragments that aremore similar, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, or 100% are highly preferred. Fragments that are 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, and 75% are also contemplated.

Preferred polypeptides may also be described as having an amino acidsequence encoded by a nucleic acid sequence at least 80% identical to afragment of SEQ ID NO:3 encoding a ligand binding fragment of VEGFR-2.Nucleic acid fragments that are more similar, e.g., 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% are highly preferred.Fragments that are 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75% arealso contemplated. A genus of similar polypeptides can alternatively bedefined by the ability of encoding polynucleotides to hybridize to thecomplement of a nucleotide sequence that corresponds to the cDNAsequence encoding the R-2 receptor. For example, a preferred bindingunit polypeptide comprises an amino acid sequence that binds one or moreR-2 ligands and that is encoded by a nucleotide sequence that hybridizesto the complement of SEQ ID NO: 3 under moderately or highly stringentconditions discussed herein.

Exemplary R2 fragments for use as binding unit polypeptides (or for useas a starting point for designing R-2 analogs) have an amino terminalresidue selected from the group consisting of positions 1 to 118 of SEQID NO: 4, and a carboxy terminal residue selected from the groupconsisting of positions 326 to 764 of SEQ ID NO: 4, wherein VEGFR-2fragment binds at least one of VEGF-A, VEGF-C, VEGF-D, and VEGF-E.Exemplary R2 fragments for use as binding unit polypeptides (or for useas a starting point for designing R-2 analogs) may alternatively have anamino terminal residue selected from the group consisting of positions 1to 192 of SEQ ID NO: 4, and a carboxy terminal residue selected from thegroup consisting of positions 393 to 764 of SEQ ID NO: 4, wherein theVEGFR-2 fragment binds at least one of VEGF-A, VEGF-C, VEGF-D, andVEGF-E. Exemplary R2 fragments for use as binding unit polypeptides (orfor use as a starting point for designing R-2 analogs) may also have anamino terminal residue selected from the group consisting of positions 1to 48 of SEQ ID NO: 4, and a carboxy terminal residue selected from thegroup consisting of positions 214 to 764 of SEQ ID NO: 4, wherein theVEGFR-2 fragment binds at least one of VEGF-A, VEGF-C, VEGF-D, andVEGF-E.

In some embodiments, a binding unit of the binding construct comprises afragment of R-2, SEQ ID NO: 4, selected from the group consisting ofpositions 24-326 (SEQ ID NO: 8), 118-326 (SEQ ID NO: 20), positions118-220 (SEQ ID NO: 22), positions 118-226 (SEQ ID NO: 24), andpositions 118-232 (SEQ ID NO: 26). In some embodiments, a binding unitof the binding construct comprises a fragment of R-2, SEQ ID NO: 4,selected from the group consisting of positions 106-240, positions112-234, positions 114-220, positions 115-220, positions 116-222,positions 117-220, positions 118-221, positions 118-222, positions118-223, positions 118-224, and positions 118-228. In some embodiments,a binding unit of the binding construct comprises a fragment of R-2, SEQID NO: 4, selected from the group consisting of positions 48-203, and145-310 and 48-310. Exemplary embodiments are also discussed in Example1.

3. VEGFR-3-Derived Binding Units

In some embodiments, a binding unit comprises a polypeptide similar oridentical in amino acid sequence to a VEGFR-3 polypeptide or fragmentthereof, preferably from the same species as the targeted growthfactor(s). Thus, for binding to human growth factors, a binding unitpreferably comprises a polypeptide that comprises an amino acid similaror identical to a fragment of SEQ ID NO: 6, where the fragment and thepolypeptide binds one or more growth factors selected from the groupconsisting of VEGF-C and VEGF-D. The fragment minimally comprises enoughof the VEGFR-3 sequence to bind the ligand, and may comprise thecomplete receptor. Extracellular domain fragments are preferred.Preferred polypeptides have an amino acid sequence at least 80%identical to a ligand binding fragment thereof. Fragments that are moresimilar, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, or 100% are highly preferred. Fragments that are 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, and 75% are also contemplated. A genus ofsimilar polypeptides can alternatively be defined by the ability ofencoding polynucleotides to hybridize to the complement of a nucleotidesequence that corresponds to the cDNA sequence encoding the R-3receptor.

Preferred polypeptides may also be described as having an amino acidsequence encoded by a nucleic acid sequence at least 80% identical to afragment of SEQ ID NO:5 encoding a ligand binding fragment of VEGFR-3.Nucleic acid fragments that are more similar, e.g., 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% are highly preferred.Fragments that are 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75% arealso contemplated. For example, a preferred binding unit polypeptidecomprises an amino acid sequence that binds one or more R-3 ligands andthat is encoded by a nucleotide sequence that hybridizes to thecomplement of SEQ ID NO: 5 under moderately or highly stringentconditions discussed herein.

Exemplary R-3 fragments for use as binding unit polypeptides (or for useas a starting point for designing R-3 analogs) have an amino terminalresidue selected from the group consisting of positions 1 to 47 of SEQID NO: 6, and a carboxy terminal residue selected from the groupconsisting of positions 226 to 775 of SEQ ID NO: 6, wherein VEGFR-3fragment binds at least one of VEGF-C and VEGF-D.

In some embodiments, a binding unit of the binding construct comprises afragment of R-3, SEQ ID NO: 6, selected from the group consisting ofpositions 1-226 (SEQ ID NO: 38), positions 1-229 (SEQ ID NO: 36), andpositions 1-329 (SEQ ID NO: 44). In some embodiments, a binding unit ofthe binding construct comprises a fragment of R-3, SEQ ID NO: 6,selected from the group consisting of positions 47-224, positions47-225, positions 47-226, positions 47-227, positions 47-228, positions47-229, positions 47-230, positions 47-231, positions 47-232, positions47-236, positions 47-240, and positions 47-245. In some embodiments, abinding unit of the binding construct comprises a fragment of R-3, SEQID NO: 6, selected from the group consisting of positions 47-314,positions 47-210, and positions 47-247. Exemplary embodiments are alsodiscussed in Example 1.

4. Neuropilin-1-Derived Binding Units

In some embodiments, a binding unit comprises a polypeptide similar oridentical in amino acid sequence to a neuropilin-1 polypeptide orfragment thereof, preferably from the same species as the targetedgrowth factor(s). Thus, for binding to human growth factors, a bindingunit preferably comprises a polypeptide that comprises an amino acidsimilar or identical to a fragment of SEQ ID NO: 113, where the fragmentand the polypeptide binds one or more growth factors selected from thegroup consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-E, and PlGF. Thefragment minimally comprises enough of the neuropilin-1 sequence to bindthe ligand, and may comprise the complete receptor. Extracellular domainfragments are preferred. Preferred polypeptides have an amino acidsequence at least 80% identical to a ligand binding fragment thereof.Fragments that are more similar, e.g., 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5%, or 100% are highly preferred. Fragmentsthat are 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75% are alsocontemplated.

Preferred polypeptides may also be described as having an amino acidsequence encoded by a nucleic acid sequence at least 80% identical to afragment of SEQ ID NO: 112 encoding a ligand binding fragment ofneuropilin-1. Nucleic acid fragments that are more similar, e.g., 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% arehighly preferred. Fragments that are 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, and 75% are also contemplated. A genus of similar polypeptides canalternatively be defined by the ability of encoding polynucleotides tohybridize to the complement of a nucleotide sequence that corresponds tothe cDNA sequence encoding the neuropilin-1 receptor. For example, apreferred binding unit polypeptide comprises an amino acid sequence thatbinds one or more neuropilin-1 ligands and that is encoded by anucleotide sequence that hybridizes to the complement of SEQ ID NO: 112under moderately or highly stringent conditions discussed herein.

Exemplary neuropilin-1 fragments for use as binding unit polypeptides(or for use as a starting point for designing neuropilin-1 analogs)comprise a neuropilin-1 extracellular domain amino acid sequencecomprising residues 22-856 of SEQ ID NO: 113, or a portion thereof;wherein the neuropilin-1 fragment and the binding unit bind at least onegrowth factor selected from the group consisting of VEGF-A, VEGF-B,VEGF-C, VEGF-E, and PlGF.

5. Neuropilin-2-Derived Binding Units

In some embodiments, a binding unit comprises a polypeptide similar oridentical in amino acid sequence to a neuropilin-2 polypeptide orfragment thereof, preferably from the same species as the targetedgrowth factor(s). Thus, for binding to human growth factors, a bindingunit preferably comprises a polypeptide that comprises an amino acidsimilar or identical to a fragment of SEQ ID NO: 115, wherein thefragment and the polypeptide binds one or more growth factors selectedfrom the group consisting of VEGF-A, VEGF-C, and PlGF. The fragmentminimally comprises enough of the neuropilin-2 sequence to bind theligand, and may comprise the complete receptor. Extracellular domainfragments are preferred. Preferred polypeptides have an amino acidsequence at least 80% identical to a ligand binding fragment thereof.Fragments that are more similar, e.g., 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5%, or 100% are highly preferred. Fragmentsthat are 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75% are alsocontemplated.

Preferred polypeptides may also be described as having an amino acidsequence encoded by a nucleic acid sequence at least 80% identical to afragment of SEQ ID NO: 114 encoding a ligand binding fragment ofneuropilin-2. Nucleic acid fragments that are more similar, e.g., 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% arehighly preferred. Fragments that are 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, and 75% are also contemplated. A genus of similar polypeptides canalternatively be defined by the ability of encoding polynucleotides tohybridize to the complement of a nucleotide sequence that corresponds tothe cDNA sequence encoding the neuropilin-2 receptor. For example, apreferred binding unit polypeptide comprises an amino acid sequence thatbinds one or more neuropilin-2 ligands and that is encoded by anucleotide sequence that hybridizes to the complement of SEQ ID NO: 114under moderately or highly stringent conditions discussed herein.

Exemplary neuropilin-2 fragments for use as binding unit polypeptidescomprising residues 21-864 of SEQ ID NO: 115, or a portion thereof;wherein the neuropilin-2 fragment and the binding unit bind at least onegrowth factor selected from the group consisting of VEGF-A, VEGF-C, andPlGF.

Further neuropilin-1 and -2 species, isoforms, soluble fragments, etc.,are provided in WO03/029814, U.S. application Ser. Nos. 10/262,538,10/669,176, and 60/505,607, which are incorporated by reference in theirentireties.

6. PDGFR-Alpha-Derived Binding Units

In some embodiments, a binding unit comprises a polypeptide similar oridentical in amino acid sequence to a PDGFR-α polypeptide or fragmentthereof, preferably from the same species as the targeted growthfactor(s). Thus, for binding to human growth factors, a binding unitpreferably comprises a polypeptide that comprises an amino acid similaror identical to a fragment of SEQ ID NO: 117, where the fragment and thepolypeptide binds one or more growth factors selected from the groupconsisting of PDGF-A, PDGF-B, and PDGF-C. The fragment minimallycomprises enough of the PDGFR-α sequence to bind the ligand, and maycomprise the complete receptor. Extracellular domain fragments arepreferred. Preferred polypeptides have an amino acid sequence at least80% identical to a ligand binding fragment thereof. Fragments that aremore similar, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, or 100% are highly preferred. Fragments that are 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, and 75% are also contemplated. A genus ofsimilar polypeptides can alternatively be defined by the ability ofencoding polynucleotides to hybridize to the complement of a nucleotidesequence that corresponds to the cDNA sequence encoding the R-αreceptor.

Preferred polypeptides may also be described as having an amino acidsequence encoded by a nucleic acid sequence at least 80% identical to afragment of SEQ ID NO: 116 encoding a ligand binding fragment of R-α.Nucleic acid fragments that are more similar, e.g., 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% are highly preferred.Fragments that are 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75% arealso contemplated. For example, a preferred binding unit polypeptidecomprises an amino acid sequence that binds one or more R-α ligands andthat is encoded by a nucleotide sequence that hybridizes to thecomplement of SEQ ID NO: 116 under moderately or highly stringentconditions discussed herein.

Exemplary R-α fragments for use as binding unit polypeptides (or for useas a starting point for designing R-α analogs) have an amino terminalresidue selected from the group consisting of positions 1 to 123 of SEQID NO: 117, and a carboxy terminal residue selected from the groupconsisting of positions 313 to 524 of SEQ ID NO: 117, wherein thePDGFR-α fragment binds at least one of PDGF-A, PDGF-B, and PDGF-C.

7. PDGFR-Beta-Derived Binding Units

In some embodiments, a binding unit comprises a polypeptide similar oridentical in amino acid sequence to a R-β polypeptide or fragmentthereof, preferably from the same species as the targeted growthfactor(s). Thus, for binding to human growth factors, a binding unitpreferably comprises a polypeptide that comprises an amino acid similaror identical to a fragment of SEQ ID NO: 119, where the fragment and thepolypeptide binds one or more growth factors selected from the groupconsisting of PDGF-B and PDGF-D. The fragment minimally comprises enoughof the PDGFR-β sequence to bind the ligand, and may comprise thecomplete receptor. Extracellular domain fragments are preferred.Preferred polypeptides have an amino acid sequence at least 80%identical to a ligand binding fragment thereof. Fragments that are moresimilar, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, or 100% are highly preferred. Fragments that are 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, and 75% are also contemplated. A genus ofsimilar polypeptides can alternatively be defined by the ability ofencoding polynucleotides to hybridize to the complement of a nucleotidesequence that corresponds to the cDNA sequence encoding the R-βreceptor.

Preferred polypeptides may also be described as having an amino acidsequence encoded by a nucleic acid sequence at least 80% identical to afragment of SEQ ID NO: 118 encoding a ligand binding fragment ofPDGFR-β. Nucleic acid fragments that are more similar, e.g., 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% are highlypreferred. Fragments that are 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,and 75% are also contemplated. For example, a preferred binding unitpolypeptide comprises an amino acid sequence that binds one or more R-βligands and that is encoded by a nucleotide sequence that hybridizes tothe complement of SEQ ID NO: 118 under moderately or highly stringentconditions discussed herein.

Exemplary R-β fragments for use as binding unit polypeptides (or for useas a starting point for designing R-β analogs) have an amino terminalresidue selected from the group consisting of positions 1 to 124 of SEQID NO: 119, and a carboxy terminal residue selected from the groupconsisting of positions 314 to 531 of SEQ ID NO: 119, wherein PDGFR-βfragment binds at least one of PDGF-B and PDGF-D.

8. Other Binding Units

Although a binding unit may comprise a polypeptide similar or identicalto an extracellular domain fragment of a growth factor receptor tyrosinekinase, other binding units are contemplated as well. In someembodiments, the binding unit is generated using phage display. In someembodiments, the binding unit comprises an antibody. In someembodiments, a binding unit comprises a polypeptide comprising anantibody (antigen binding) fragment, e.g., a domain antibody. Bindingunits, as well as binding constructs, need not comprise a polypeptide.In some embodiments, the binding construct comprises nucleic acid, e.g.,DNA or RNA, such as an aptamer. In some embodiments, the bindingconstruct comprises polysaccharides.

Growth factor binding molecules that have been described in theliterature may be used as binding units to construct binding constructsof the inventory including molecules taught by the following: Veikkola,T., et al., Cancer Res. 60:203-212 (2000); Davis-Smyth, T., et al., EMBOJ., 15(18): 4919-27 (1996), U.S. Pat. Nos. 5,952,199; 6,100,071;6,383,486; U.S. Pat. Appl. Nos. 20030092604; Niwa, et al, U.S. Pat. No.6,348,333; Fairbrother, et al., Biochemistry, 37:17754-64 (1998);Starovasnik, M. et al., J. Mol. Biol., 293: 531-44 (1999); Wiesmann, C.,et al., Cell, 91:695-704 (1997); Fuh, et al., J. Biol. Chem., 273(18):11197-11204 (1998); Shinkai, A. et al., J. Biol. Chem., 273(47):31283-88(1998); Lu, et al., J. Biol. Chem., 275(19): 14321-14330 (2000); Lu etal., J. Immunological Methods, 230:159-71 (1999); Lu, et al., J. Biol.Chem., 278(44): 43496-43507 (2003); Makkinen, T., et al., NatureMedicine, 7(2), 199-205 (2001); Alitalo, et al., WO 02/060950; Karpanen,T., et al., Cancer Research 61:1786-90 (2001); Liu, et al., U.S. Pat.Appl. Publ. No. 2003/0064053; Kubo, H., et al., Blood, 96(2): 546-553(2000); Rosen, Hematol. Oncol. Clin. N. Am., 16:1173-1187 (2002);Kaplan, et al., Growth Factors, 14:243-256 (1997); Thomas, et al., U.S.Pat. No. 6,375,929; Kendall and Thomas, PNAS, U.S.A., 90:10705-10709(1993); Kovesdi, U.S. Pat. Appl. Publ. No. 2003/0053989; Daly, et al.,U.S. Pat. Appl. Publ. No.: 2004/0014667; and Lokker, et al., J. Biol.Chem. 272: 33037-33044 (1997). These and other documents cited in thisapplication are incorporated in their entireties. Molecules that havenot previously been tested for their ability to bind to a particulargrowth factor may tested according to the assays provided herein. Forexample, some of the above documents teach a R-2 fragment that bindsVEGF-A. That same molecule may be tested for its ability to bind VEGF-C.

Except as otherwise noted, descriptions supplied for receptors, alsoapply to receptor fragments and such fragments incorporated into bindingconstructs as described herein.

The growth factor receptors, from which binding units may be derived,include splice variants and naturally-occurring allelic variations.Allelic variants are well known in the art, and represent alternativeforms or a nucleic acid sequence that comprise substitution, deletion oraddition of one or more nucleotides, but which do not result in anysubstantial functional alteration of the encoded polypeptide. Standardmethods can readily be used to generate such polypeptides includingsite-directed mutagenesis of polynucleotides, or specific enzymaticcleavage and ligation. Similarly, use of peptidomimetic compounds orcompounds in which one or more amino acid residues are replaced by anon-naturally-occurring amino acid or an amino acid analog that retainbinding activity is contemplated. Preferably, where amino acidsubstitution is used, the substitution is conservative, i.e. an aminoacid is replaced by one of similar size and with similar chargeproperties. As used herein, the term “conservative substitution” denotesthe replacement of an amino acid residue by another, biologicallysimilar residue. Examples of conservative substitutions include thesubstitution of one hydrophobic residue such as isoleucine, valine,leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan,tyrosine, norleucine or methionine for another, or the substitution ofone polar residue for another, such as the substitution of arginine forlysine, glutamic acid for aspartic acid, or glutamine for asparagine,and the like. Neutral hydrophilic amino acids that can be substitutedfor one another include asparagine, glutamine, serine and threonine. Theterm “conservative substitution” also includes the use of a substitutedamino acid in place of an unsubstituted amino acid.

Alternatively, conservative amino acids can be grouped as described inLehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY,pp. 71-77 (1975)) as set out in the following:

Non-polar (hydrophobic)

-   -   A. Aliphatic: A, L, I, V, P,    -   B. Aromatic: F, W,    -   C. Sulfur-containing: M,    -   D. Borderline: G.

Uncharged-polar

-   -   A. Hydroxyl: S, T, Y,    -   B. Amides: N, Q,    -   C. Sulfhydryl: C,    -   D. Borderline: G.

Positively Charged (Basic): K, R, H.

Negatively Charged (Acidic): D, E.

B. Linkers

While binding units may be directly attached to one another (via apeptide, disulfide or other type of covalent bond), the bindingconstructs of the present invention may further comprise a (one or more)linker that connects together two or more different binding units, e.g.,a receptor fragments with another receptor fragment, or even a copy ofitself. A linker may also link a binding unit to other substituentsdescribed herein. The linker is generally a heterologous proteinpolypeptide. In some embodiments, the linker comprises a peptide thatlinks the binding units to form a single continuous peptide that can beexpressed as a single molecule. Linkers may be chosen such that they areless likely to induce an allergic reaction. Polysaccharides or othermoieties also may be used to link binding units to form a bindingconstruct.

More than one linker may be used per binding construct. The linker maybe selected for optimal conformational (steric) freedom between thevarious ligand binding units to allow them to interact with each otherif desired, e.g., to form dimers, or to allow them to interact withligand. The linker may be linear such that consecutive binding units arelinked in series, or the linker may serve as a scaffold to which variousbinding units are attached, e.g., a branched linker. A linker may alsohave multiple branches, e.g., as disclosed in Tam, J. Immunol. Methods196:17 (1996). Binding units may be attached to each other or to thelinker scaffold via N-terminal amino groups, C-terminal carboxyl groups,side chains, chemically modified groups, side chains, or other means.

Linker peptides may be designed to have sequences that permit desiredcharacteristics. For example, the use of glycyl residues allow for arelatively large degree of conformational freedom, whereas a prolinewould tend to have the opposite effect. Peptide linkers may be chosen sothat they achieve particular secondary and tertiary structures, e.g.,alpha helices, beta sheets or beta barrels. Quaternary structure canalso be utilized to create linkers that join two binding units togethernon-covalently. For example, fusing a protein domain with a hydrophobicface to each binding unit may permit the joining of the two bindingunits via the interaction between the hydrophobic interaction of the twomolecules. In some embodiments, the linker may provide for polarinteractions. For example, a leucine zipper domain of theproto-oncoproteins Myc and Max, respectively, may be used. Luscher andLarsson, Ongogene 18:2955-2966 (1999). In some embodiments, the linkerallows for the formation of a salt bridge or disulfide bond. Linkers maycomprise non-naturally occurring amino acids, as well as naturallyoccurring amino acids that are not naturally incorporated into apolypeptide. In some embodiments, the linker comprises a coordinationcomplex between a metal or other ion and various residues from themultiple peptides joined thereby.

Linear peptide linkers of at least one amino acid residue arecontemplated. In some embodiments the linker has more than 10,000residues. In some embodiments the linker has from 1-10,000 residues. Insome embodiments, the linker has from 1-1000 residues. In someembodiments, the linker has from 1-100 residues. In some embodiments,the linker has from 1-50 residues. In some embodiments the linker has1-10 residues. In some embodiments, the linear peptide linker comprisesresidues with relatively inert side chains. Peptide linker amino acidresidues need not be linked entirely or at all via alpha-carboxy andalpha-amino groups. That is, peptides may be linked via side chaingroups of various residues.

The linker may affect whether the polypeptide(s) to which it is fused tois able to dimerize to each other or to another polypeptide. The linkerserves a number of functions. Native receptor monomers restrained to theroughly two-dimensional plane of the cell membrane enjoy a relativelyhigh local concentration and in the availability of co-receptors(binding units), increasing the probability of finding a partner.Receptors free in solution lacking such advantages may be aided by alinker that increases the effective concentration of the monomers.

In some embodiments, a binding construct may comprise more than one typeof linker. Suitable linkers may also comprise the chemical modificationsdiscussed below.

C. Substituents And Other Chemical Modifications

The binding constructs of the invention may be chemically modified withvarious substituents. Such modifications preferably does notsubstantially reduce the growth factor binding affinities orspecificities of the binding construct. Rather, the chemicalmodifications impart additional desirable characteristics as discussedherein. Chemical modifications may take a number of different forms suchas heterologous peptides, polysaccarides, lipids, radioisotopes,non-standard amino acid resides and nucleic acids, metal chelates, andvarious toxins.

The receptor fragments, binding constructs, and other peptide moleculesof the present invention may be fused to heterologous peptides to confervarious properties, e.g., increased solubility, modulation of clearance,targeting to particular cell or tissue types. In some embodiments, thereceptor fragment is linked to a Fc domain of IgG or otherimmunoglobulin. In some embodiments, a receptor fragment is fused toalkaline phosphatase (AP). Methods for making Fc or AP fusion constructsare found in WO 02/060950. By fusing the ligand binding domain ofVEGFR-2 or VEGFR-3 (or other receptors) with protein domains that havespecific properties (e.g. half life, bioavailability, interactionpartners) it is possible to confer these properties to the VEGFR bindingdomains (e.g., the receptor binding domain could be engineered to have aspecific tissue distribution or specific biological half life). In someembodiments, binding construct may include a co-receptor and a VEGFRfragment.

The particular heterologous polypeptide used in a particular constructcan influence whether or not a growth factor receptor fragment willdimerize, which in turn may affect ligand binding. Fc fusion all maypermit dimers, whereas AP fusions may permit monomers, cited, whichalong with Ig-domain boundary differences as possible reasons fordifferent results obtained by different groups for receptor fragmentsbinging to ligands. [Lu, et al., J. Biol. Chem. 275(19): 14321-14330(2000).]

For substituents such as an Fc region of human IgG, the fusion can befused directly to a binding construct or fused through an interveningsequence. For example, a human IgG hinge, CH2 and CH3 region may befused at either the N-terminus or C-terminus of a binding construct toattach the Fc region. The resulting Fc-fusion construct enablespurification via a Protein A affinity column (Pierce, Rockford, Ill.).Peptide and proteins fused to an Fc region can exhibit a substantiallygreater half-life in vivo than the unfused counterpart. A fusion to anFc region allows for dimerization/multimerization of the fusionpolypeptide. The Fc region may be a naturally occurring Fc region, ormay be modified for superior characteristics, e.g., therapeuticqualities, circulation time, reduced aggregation.

Polypeptides can be modified, for instance, by glycosylation, amidation,carboxylation, or phosphorylation, or by the creation of acid additionsalts, amides, esters, in particular C-terminal esters, and N-acylderivatives. The proteins also can be modified to create peptidederivatives by forming covalent or noncovalent complexes with othermoieties. Covalently bound complexes can be prepared by linking thechemical moieties to functional groups on the side chains of amino acidscomprising the peptides, or at the N- or C-terminus.

Polypeptides can be conjugated to a reporter group, including, but notlimited to a radiolabel, a fluorescent label, an enzyme (e.g., thatcatalyzes a calorimetric or fluorometric reaction), a substrate, a solidmatrix, or a carrier (e.g., biotin or avidin). Examples of analogs aredescribed in WO 98/28621 and in Olofsson, et al., Proc. Nat'l. Acad.Sci. USA, 95:11709-11714 (1998), U.S. Pat. Nos. 5,512,545, and5,474,982; U.S. Patent Application Nos. 20020164687 and 20020164710.

Cysteinyl residues most commonly are reacted with haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carbocyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol,orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic orcarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylissurea; 2,4 pentanedione; and transaminase catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using 125I or 131I toprepare labeled proteins for use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R1) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking thebinding construct to water-insoluble support matrixes. Such derivationmay also provide the linker that may connect adjacent binding elementsin a binding construct, or a binding elements to a heterologous peptide,e.g., a Fc fragment. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homo-bifunctional imidoesters, including disuccinimidyl esterssuch as 3,3′-dithiiobis(succinimidylpropioonate), and bifunctionalmaleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents suchas methyl-3-[(p-azidophenyl) dithio] propioimidate yieldphotoactivatable intermediates that are capable of forming cross linksin the presence of light. Alternatively, reactive water-insolublematrices such as cyanogen bromide-activated carbohydrates and thereactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; and 4,330,440, incorporated herein byreference, are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W.H. Freeman & Co., San Francisco, pp. 79-86.1983),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups. Such derivatives are chemicallymodified polypeptide compositions in which the binding constructpolypeptide is linked to a polymer. The polymer selected is typicallywater soluble so that the protein to which it is attached does notprecipitate in an aqueous environment, such as a physiologicalenvironment. The polymer selected is usually modified to have a singlereactive group, such as an active ester for acylation or an aldehyde foralkylation, so that the degree of polymerization may be controlled asprovided for in the present methods. The polymer may be of any molecularweight, and may be branched or unbranched. Included within the scope ofthe binding construct polypeptide polymers is a mixture of polymers.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The polymers each may be of any molecular weight and may be branched orunbranched. The polymers each typically have an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of each polymer is between about 5 kDa and about 50kDa, more preferably between about 12 kDa to about 40 kDa and mostpreferably between about 20 kDa to about 35 kDa.

Suitable water soluble polymers or mixtures thereof include, but are notlimited to, N-linked or O-linked carbohydrates, sugars, phosphates,carbohydrates; sugars; phosphates; polyethylene glycol (PEG) (includingthe forms of PEG that have been used to derivatize proteins, includingmono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol);monomethoxy-polyethylene glycol; dextran (such as low molecular weightdextran, of, for example about 6 kD), cellulose; cellulose; othercarbohydrate-based polymers, poly-(N-vinyl pyrrolidone)polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol. Also encompassed by the present invention arebifunctional crosslinking molecules which may be used to preparecovalently attached multimers.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.Methods for preparing chemical derivatives of polypeptides willgenerally comprise the steps of (a) reacting the polypeptide with theactivated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby the bindingconstruct becomes attached to one or more polymer molecules, and (b)obtaining the reaction product(s). The optimal reaction conditions willbe determined based on known parameters and the desired result. Forexample, the larger the ratio of polymer molecules:protein, the greaterthe amount of attached polymer molecule. In one embodiment, the bindingconstruct polypeptide derivative may have a single polymer moleculemoiety at the amino terminus. (See, e.g., U.S. Pat. No. 5,234,784).

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol (PEG). As used herein, polyethylene glycol is meantto encompass any of the forms of PEG that can be used to derivatizeother proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethyleneglycol. PEG is a linear or branched neutral polyether, available in abroad range of molecular weights, and is soluble in water and mostorganic solvents. PEG is effective at excluding other polymers orpeptides when present in water, primarily through its high dynamic chainmobility and hydrophibic nature, thus creating a water shell orhydration sphere when attached to other proteins or polymer surfaces.PEG is nontoxic, non-immunogenic, and approved by the Food and DrugAdministration for internal consumption.

Proteins or enzymes when conjugated to PEG have demonstratedbioactivity, non-antigenic properties, and decreased clearance rateswhen administered in animals. F. M. Veronese et al., Preparation andProperties of Monomethoxypoly(ethylene glycol)-modified Enzymes forTherapeutic Applications, in J. M. Harris ed., Poly(Ethylene Glycol)Chemistry—Biotechnical and Biomedical Applications, 127-36, 1992,incorporated herein by reference. These phenomena are due to theexclusion properties of PEG in preventing recognition by the immunesystem. In addition, PEG has been widely used in surface modificationprocedures to decrease protein adsorption and improve bloodcompatibility. S. W. Kim et al., Ann. N.Y. Acad. Sci. 516: 116-30 1987;Jacobs et al., Artif Organs 12: 500-501, 1988; Park et al., J. Poly.Sci, Part A 29:1725-31, 1991, incorporated herein by reference.Hydrophobic polymer surfaces, such as polyurethanes and polystyrene canbe modified by the grafting of PEG (MW 3,400) and employed asnonthrombogenic surfaces. Surface properties (contact angle) can be moreconsistent with hydrophilic surfaces, due to the hydrating effect ofPEG. More importantly, protein (albumin and other plasma proteins)adsorption can be greatly reduced, resulting from the high chainmotility, hydration sphere, and protein exclusion properties of PEG.

PEG (MW 3,400) was determined as an optimal size in surfaceimmobilization studies, Park et al., J. Biomed. Mat. Res. 26:739-45,1992, while PEG (MW 5,000) was most beneficial in decreasing proteinantigenicity. (F. M. Veronese et al., In J. M. Harris, et al.,Poly(Ethylene Glycol) Chemistry—Biotechnical and BiomedicalApplications, 127-36.)

Methods for preparing pegylated binding construct polypeptides willgenerally comprise the steps of (a) reacting the polypeptide withpolyethylene glycol (such as a reactive ester or aldehyde derivative ofPEG) under conditions whereby the binding construct polypeptide becomesattached to one or more PEG groups, and (b) obtaining the reactionproduct(s). In general, the optimal reaction conditions for theacylation reactions will be determined based on known parameters and thedesired result. For example, the larger the ratio of PEG: protein, thegreater the percentage of poly-pegylated product. In some embodiments,the binding construct will have a single PEG moiety at the N-terminus.See U.S. Pat. No. 8,234,784, herein incorporated by reference.

Derivatized binding constructs disclosed herein may have additionalactivities, enhanced or reduced biological activity, or othercharacteristics, such as increased or decreased half-life, as comparedto the non-derivatized molecules.

II. POLYNUCLEOTIDES ENCODING BINDING CONSTRUCTS AND EXPRESSION SYSTEMS

The invention comprises not only the binding constructs, binding units,and polypeptides described herein, and uses thereof, but also nucleicacids encoding such molecules, vectors comprising such molecules, andhost cells comprising such vectors, and uses thereof. Methods employingany of the constructs, units, polypeptides, nucleic acids, vectors, andhosts cells for the therapeutic uses described herein are all consideredaspects of the invention.

A. Nucleic Acids of the Invention

This invention also includes nucleic acid molecules whose sequenceencode the polypeptides, binding units, and binding constructs, for usein compositions and methods of the invention. Nucleic acid moleculesinclude those molecules which comprise nucleotide sequences whichhybridize under moderately or highly stringent conditions as definedherein with the fully complementary sequence of the nucleic acidmolecule of receptor tyrosine kinases described in Table 1A, or of amolecule encoding a polypeptide, which polypeptide comprises thereceptor tyrosine kinase amino acids sequences described in Table 1A, orof a nucleic acid fragment as defined herein, or of a nucleic acidfragment encoding a polypeptide as defined herein.

Hybridization probes may be prepared using the sequences provided hereinto screen cDNA, genomic or synthetic DNA libraries for relatedsequences. Regions of the DNA and/or amino acid sequence that exhibitsignificant identity to known sequences are readily determined usingsequence alignment algorithms as described herein, and those regions maybe used to design probes for screening.

The term “highly stringent conditions” refers to those conditions thatare designed to permit hybridization of DNA strands whose sequences arehighly complementary, and to exclude hybridization of significantlymismatched DNAs. Hybridization stringency is principally determined bytemperature, ionic strength, and the concentration of denaturing agentssuch as formamide. Examples of “highly stringent conditions” forhybridization and washing are 0.015 M sodium chloride, 0.0015 M sodiumcitrate at 65-68° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 50% formamide at 42° C. See Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, (Cold Spring Harbor, N.Y. 1989); and Anderson et al.,Nucleic Acid Hybridization: a Practical approach, Ch. 4, IRL PressLimited (Oxford, England). Limited, Oxford, England. Other agents may beincluded in the hybridization and washing buffers for the purpose ofreducing non-specific and/or background hybridization. Examples are 0.1%bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodiumpyrophosphate, 0.1% sodium dodecylsulfate (NaDodSO₄ or SDS), ficoll,Denhardt's solution, sonicated salmon sperm DNA (or anothernon-complementary DNA), and dextran sulfate, although other suitableagents can also be used. The concentration and types of these additivescan be changed without substantially affecting the stringency of thehybridization conditions. Hybridization experiments are usually carriedout at pH 6.8-7,4,6,8-7.4; however, at typical ionic strengthconditions, the rate of hybridization is nearly independent of pH. SeeAnderson et al., Nucleic Acid Hybridization: a Practical Approach, Ch.4, IRL Press Limited (Oxford, England).

Factors affecting the stability of a DNA duplex include basecomposition, length, and degree of base pair mismatch. Hybridizationconditions can be adjusted by one skilled in the art in order toaccommodate these variables and allow DNAs of different sequencerelatedness to form hybrids. The melting temperature of a perfectlymatched DNA duplex can be estimated by the following equation:

T _(m)(° C.)=81.5+16.6(log [Na⁺])+0.41(% G+C)−600/N−0.72(% formamide)

where N is the length of the duplex formed, [Na⁺] is the molarconcentration of the sodium ion in the hybridization or washingsolution, % G+C is the percentage of (guanine+cytosine) bases in thehybrid. For imperfectly matched hybrids, the melting temperature isreduced by approximately 1° C. for each 1% mismatch.

The term “moderately” stringent conditions” refers to conditions underwhich a DNA duplex with a greater degree of base pair mismatching thancould occur under “highly stringent conditions” is able to form.Examples of typical “moderately stringent conditions” are 0.015 M sodiumchloride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodiumchloride, 0.0015 M sodium citrate, and 20% formamide at 37-50° C. By wayof example, a “moderately stringent” condition of 50° C. in 0.015 Msodium ion will allow about a 21% mismatch.

It will be appreciated by those skilled in the art that there is noabsolute distinction between “highly” and “moderately” stringentconditions. For example, at 0.015M sodium ion (no formamide), themelting temperature of perfectly matched long DNA is about 71° C. With awash at 65° C. (at the same ionic strength), this would allow forapproximately a 6% mismatch. To capture more distantly relatedsequences, one skilled in the art can simply lower the temperature orraise the ionic strength.

A good estimate of the melting temperature in 1M NaCl* foroligonucleotide probes up to about 20 nt is given by:

Tm=2C per A−T base pair+4° C. per G−C base pair

*The sodium ion concentration in 6× salt sodium citrate (SSC) is 1 M.See Suggs et al., Developmental Biology Using Purified Genes, p. 683,Brown and Fox (eds.) (1981).

High stringency washing conditions for oligonucleotides are usually at atemperature of 0-5° C. below the Tm of the oligonucleotide in 6×SSC,0.1% SDS.

Differences in the nucleic acid sequence may result in conservativeand/or non-conservative modifications of the amino acid sequencerelative to the amino acid sequence. The invention is also directed toan isolated and/or purified DNA that corresponds to, or that hybridizesunder stringent conditions with, any one of the foregoing DNA sequences.

B. Preparation of DNA Encoding Ligand, Receptor, and Binding ConstructPolypeptides

A nucleic acid molecule encoding all or part of a polypeptide of theinvention such as a binding construct or binding unit of the inventioncan be made in a variety of ways, including, without limitation,chemical synthesis, cDNA or genomic library screening, expressionlibrary screening, and/or PCR amplification of cDNA or genomic DNA.These methods and others useful for isolating such DNA are set forth,for example, by Sambrook, et al., “Molecular Cloning: A LaboratoryManual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989), by Ausubel, et al., eds., “Current Protocols In MolecularBiology,” Current Protocols Press (1994), and by Berger and Kimmel,“Methods In Enzymology: Guide To Molecular Cloning Techniques,” vol.152, Academic Press, Inc., San Diego, Calif. (1987). Preferred nucleicacid sequences are mammalian sequences, such as human, rat, and mouse.

Chemical synthesis of nucleic acid molecules can be accomplished usingmethods well known in the art, such as those set forth by Engels, etal., Angew. Chem. Intl. Ed., 28:716-734 (1989). These methods include,inter alia, the phosphotriester, phosphoramidite and H-phosphonatemethods of nucleic acid synthesis. Nucleic acids larger than about 100nucleotides in length can be synthesized as several fragments, eachfragment being up to about 100 nucleotides in length. The fragments canthen be ligated together, as described below, to form the full lengthnucleic acid of interest. A preferred method is polymer-supportedsynthesis using standard phosphoramidite chemistry.

C. Preparation of a Vector for Expression

The term “vector” refers to a nucleic acid molecule amplification,replication, and/or expression vehicle, often derived from or in theform of a plasmid or viral DNA or RNA system, where the plasmid or viralDNA or RNA is functional in a selected host cell, such as bacterial,yeast, plant, invertebrate, and/or mammalian host cells. The vector mayremain independent of host cell genomic DNA or may integrate in whole orin part with the genomic DNA. The vector will contain all necessaryelements so as to be functional in any host cell it is compatible with.Such elements are set forth below.

Nucleic acid encoding a polypeptide or fragment thereof has beenisolated, it is preferably inserted into an amplification and/orexpression vector in order to increase the copy number of the geneand/or to express the encoded polypeptide in a suitable host cell and/orto transform cells in a target organism (to express the polypeptide invivo). Numerous commercially available vectors are suitable, though“custom made” vectors may be used as well. The vector is selected to befunctional in a particular host cell or host tissue (i.e., forreplication and/or expression). The polypeptide or fragment thereof maybe amplified/expressed in prokaryotic and/or eukaryotic host cells,e.g., yeast, insect (baculovirus systems), plant, and mammalian cells.Selection of the host cell will depend at least in part on whether thepolypeptide or fragment thereof is to be glycosylated. If so, yeast,insect, or mammalian host cells are preferable; yeast and mammaliancells will glycosylate the polypeptide if a glycosylation site ispresent on the amino acid sequence.

Typically, the vectors used in any of the host cells will contain 5′flanking sequence and other regulatory elements such as an enhancer(s),a promoter, an origin of replication element, a transcriptionaltermination element, a complete intron sequence containing a donor andacceptor splice site, a signal peptide sequence, a ribosome binding siteelement, a polyadenylation sequence, a polylinker region for insertingthe nucleic acid encoding the polypeptide to be expressed, and aselectable marker element. Optionally, the vector may contain a “tag”sequence, i.e., an oligonucleotide sequence located at the 5′ or 3′ endof the coding sequence that encodes polyHis (such as hexaHis) or anothersmall immunogenic sequence. This tag will be expressed along with theprotein, and can serve as an affinity tag for purification of thepolypeptide from the host cell. Optionally, the tag can subsequently beremoved from the purified polypeptide by various means such as using aselected peptidase.

The vector/expression construct may optionally contain elements such asa 5′ flanking sequence, an origin of replication, a transcriptiontermination sequence, a selectable marker sequence, a ribosome bindingsite, a signal sequence, and one or more intron sequences. The 5′flanking sequence may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination of 5′flanking sequences from more than one source), synthetic, or it may bethe native polypeptide 5′ flanking sequence. As such, the source of the5′ flanking sequence may be any unicellular prokaryotic or eukaryoticorganism, any vertebrate or invertebrate organism, or any plant,provided that the 5′ flanking sequence is functional in, and can beactivated by, the host cell machinery.

A transcription termination element is typically located 3′ to the endof the polypeptide coding sequence and serves to terminate transcriptionof the polypeptide. Usually, the transcription termination element inprokaryotic cells is a G-C rich fragment followed by a poly T sequence.Such elements can be cloned from a library, purchased commercially aspart of a vector, and readily synthesized.

Selectable marker genes encode proteins necessary for the survival andgrowth of a host cell in a selective culture medium. Typical selectablemarker genes encode proteins that (a) confer resistance to antibioticsor other toxins, e.g., ampicillin, tetracycline, or kanamycin forprokaryotic host cells, (b) complement auxotrophic deficiencies of thecell; or (c) supply critical nutrients not available from complex media.

A ribosome binding element, commonly called the Shine-Dalgarno sequence(prokaryotes) or the Kozak sequence (eukaryotes), is necessary fortranslation initiation of mRNA. The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to besynthesized. The Shine-Dalgamo sequence is varied but is typically apolypurine (i.e., having a high A-G content). Many Shine-Dalgamosequences have been identified, each of which can be readily synthesizedusing methods set forth above.

All of the elements set forth above, as well as others useful in thisinvention, are well known to the skilled artisan and are described, forexample, in Sambrook, et al., “Molecular Cloning: A Laboratory Manual,”Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) andBerger, et al., eds., “Guide To Molecular Cloning Techniques,” AcademicPress, Inc., San Diego, Calif. (1987].

For those embodiments of the invention where the recombinant polypeptideis to be secreted, a signal sequence is preferably included to directsecretion from the cell where it is synthesized. Typically, thepolynucleotide encoding the signal sequence is positioned at the 5′ endof the coding region. Many signal sequences have been identified, andany of them that are functional in a target cell or species may be usedin conjunction with the transgene.

In many cases, gene transcription is increased by the presence of one ormore introns on the vector. The intron may be naturally-occurring,especially where the transgene is a full length or a fragment of agenomic DNA sequence. The intron may be homologous or heterologous tothe transgene and/or to the transgenic mammal into which the gene willbe inserted. The position of the intron with respect to the promoter andthe transgene is important, as the intron must be transcribed to beeffective. A preferred position for an intron is 3′ to the transcriptionstart site, and 5′ to the polyA transcription termination sequence. ForcDNA transgenes, an intron is placed on one side or the other (i.e., 5′or 3′) of the transgene coding sequence. Any intron from any source,including any viral, prokaryotic and eukaryotic (plant or animal)organisms, may be used to express the polypeptide, provided that it iscompatible with the host cell(s) into which it is inserted. Alsoincluded herein are synthetic introns. Optionally, more than one intronmay be used in the vector.

Preferred vectors for recombinant expression are those that arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII (Invitrogen Company, San Diego,Calif.), pBSII (Stratagene Company, La Jolla, Calif.), and pETL(BlueBacII; Invitrogen).

After the vector has been constructed and a nucleic acid has beeninserted into the proper site of the vector, the completed vector may beinserted into a suitable host cell for amplification and/or polypeptideexpression. Commonly used include: Prokaryotic cells such as gramnegative or gram positive bacteria, i.e., any strain of E. coli,Bacillus, Streptomyces, Saccharomyces, Salmonella, and the like;eukaryotic cells such as CHO (Chinese hamster ovary) cells; human kidney293 cells; COS-7 cells; insect cells such as Sf4, Sf5, Sf9, and Sf21 andHigh 5 (all from the Invitrogen Company, San Diego, Calif.); plant cellsand various yeast cells such as Saccharomyces and Pichia. Anytransformable or transfectable cell or cell line derived from anyorganism such as bacteria, yeast, fungi, monocot and dicot plants, plantcells, and animals are suitable.

Insertion (also referred to as “transformation” or “transfection”) ofthe vector into the selected host cell may be accomplished using suchmethods as calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method. The method selected will in partbe a function of the type of host cell to be used. These methods andother suitable methods are well known to the skilled artisan, and areset forth, for example, in Sambrook, et al., supra.

The host cells containing the vector (i.e., transformed or transfected)may be cultured using standard media well known to the skilled artisan.The media will usually contain all nutrients necessary for the growthand survival of the cells. Suitable media for culturing E. coli cellsare for example, Luria Broth (LB) and/or Terrific Broth (TB). Suitablemedia for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all ofwhich may be supplemented with serum and/or growth factors as requiredby the particular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of polypeptide produced in the host cell can be evaluatedusing standard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or binding assays.

D. Purification of Polypeptides

If the polypeptide has been designed to be secreted from the host cells,the majority of polypeptide will likely be found in the cell culturemedium. If, however, the polypeptide is not secreted from the hostcells, it will be present in the cytoplasm (for eukaryotic, grampositive bacteria, and insect host cells) or in the periplasm (for gramnegative bacteria host cells).

For intracellular polypeptides, the host cells are first disruptedmechanically or osmotically to release the cytoplasmic contents into abuffered solution. The polypeptide is then isolated from this solution.

Purification of the polypeptide from solution can be accomplished usinga variety of techniques. If the polypeptide has been synthesized suchthat it contains a tag such as hexahistidine or other small peptide ateither its carboxyl or amino terminus, it may essentially be purified ina one-step process by passing the solution through an affinity columnwhere the column matrix has a high affinity for the tag or for thepolypeptide directly (i.e., a monoclonal antibody specificallyrecognizing the polypeptide). For example, polyhistidine binds withgreat affinity and specificity to nickel, thus an affinity column ofnickel (such as the Qiagen nickel columns) can be used for purificationof the His-tagged polypeptide. (See, for example, Ausubel, et al., eds.,“Current Protocols In Molecular Biology,” Section 10.11.8, John Wiley &Sons, New York (1993)).

The strong affinity a ligand for its receptor permits affinitypurification of binding constructs, and binding constructs using anaffinity matrix comprising a complementary binding partner. Affinitychromatography may be employed, e.g., using either natural bindingpartners (e.g., a ligand when purifying a binding construct withaffinity for the same) or antibodies generated using standard procedures(e.g., immunizing a mouse, rabbit or other animal with an appropriatepolypeptide). The peptides of the present invention may be used togenerate such antibodies. Known antibodies or antibodies to known growthfactor receptors may be employed when they share an epitope with atargeted binding construct.

In addition, other well known procedures for purification can be used.Such procedures include, without limitation, ion exchangechromatography, molecular sieve chromatography, HPLC, native gelelectrophoresis in combination with gel elution, and preparativeisoelectric focusing (“Isoprime” machine/technique, Hoefer Scientific).In some cases, two or more of these techniques may be combined toachieve increased purity. Preferred methods for purification includepolyhistidine tagging and ion exchange chromatography in combinationwith preparative isoelectric focusing.

Polypeptide found in the periplasmic space of the bacteria or thecytoplasm of eukaryotic cells, the contents of the periplasm orcytoplasm, including inclusion bodies (bacteria) if the processedpolypeptide has formed such complexes, can be extracted from the hostcell using any standard technique known to the skilled artisan. Forexample, the host cells can be lysed to release the contents of theperiplasm by French press, homogenization, and/or sonication. Thehomogenate can then be centrifuged.

If the polypeptide has formed inclusion bodies in the periplasm, theinclusion bodies can often bind to the inner and/or outer cellularmembranes and thus will be found primarily in the pellet material aftercentrifugation. The pellet material can then be treated with achaotropic agent such as guanidine or urea to release, break apart, andsolubilize the inclusion bodies. The solubilized polypeptide can then beanalyzed using gel electrophoresis, immunoprecipitation or the like. Ifit is desired to isolate the polypeptide, isolation may be accomplishedusing standard methods such as those set forth below and in [Marston, etal., Meth. Enz., 182:264-275 (1990).]

III. ANTI-LIGAND AND ANTI-RECEPTOR THERAPEUTIC COMPOUNDS

Anti-ligand or anti-receptor therapies as discussed below include, butare not limited to antibody, aptamer, antisense and interference RNAtechniques and therapies.

Exemplary anti-VEGFR-3 antibodies and their production are described inU.S. Pat. Nos. ______, 6,107,046 and 6,824,777; U.S. Patent PublicationNos. 2006/0269548 and 2006/0177901; and International Patent ApplicationNo. PCT/FI95/00337 (WO 95/33772), all incorporated herein by referencein their entireties.

Exemplary VEGF-D antibodies are described, for example, in InternationalPatent Application Nos. PCT/US97/14696 and PCT/US99/31332; InternationalPublication No.: WO 0037025; U.S. Pat. Nos. 6,383,484, 6,730,489 and7,097,986; and U.S. Patent Publication Nos. 2006/0177428, 2006/0024302,2002/0123481, 2005/0282228, 2004/0141917 and 2003/0125537, allincorporated herein by reference.

Exemplary VEGF-C antibodies are described, for example, in InternationalPatent Application Nos. PCT/FI1996/000427 (WO/1997/005250) andPCT/US1998/001973 (WO/1998/033917); and U.S. Patent Publication Nos.2004/0147726, 2005/0232921, 2005/0192429, 2005/0059117, 2005/0282228,2003/0176674, and 2006/0121025, 2006/0030000, and U.S. Pat. No.6,403,088 all incorporated herein by reference.

U.S. Pat. No. 7,045,133, incorporated herein by reference, describespeptidomimetic inhibitors of VEGF-D/VEGF-C/VEGFR-3.

Exemplary anti-PDGFR antibodies and other inhibitor compounds aredescribed, for example, in U.S. Pat. Nos. 5,418,135; 5,468,468;5,620,687; 5,932,580; 6,358,954; 6,642,022; and 7,105,305, allincorporated herein by reference.

Exemplary anti-VEGFR antibodies and other inhibitor compounds aredescribed, for example, in U.S. Pat. Nos. 7,056,509; 7,052,693;6,986,890; 6,897,294; 6,887,468; 6,878,720; 6,344,339; 5,955,311;5,874,542; and 5,840,301, all incorporated herein by reference.

The following description makes specific reference to the production,testing, and use of particular anti-VEGFR-2 antibodies, asrepresentative of the many receptor and growth factor and growth factorantigens described herein. The methods described may also be readilyadapted for the production of other antibodies for use according to thepresent invention, e.g., anti-growth factor ligand antibodies andanti-receptor antibodies as binding units of the binding constructs.Such antibody-type binding units may themselves the used for practicingmethods of the invention, or form one binding unit of a more complex,multivalent binding construct. In some embodiments a binding constructhas at least one binding unit that comprising a receptor fragment and atleast one binding unit that comprises an antigen binding fragment.Antibodies directed against growth factors and receptors may also beused in combination with the binding constructs of the invention.Exemplary antibodies may be found in U.S. patent application Ser. No.11/075,400, published as U.S. Patent Publication No. 2005/0282233, andrelated, co-filed International Patent Application No.PCT/US2005/007742, published as WO 2005/087812 (Attorney Docket No.28967/39820B); and U.S. Patent Publication Nos. 2006/0177428;2006/0024302; 2004/0175730; and 2004/0141917; all applications areincorporated by reference in their entireties.

A. Therapeutic Anti-VEGFR-2 Selective VEGF-A Antagonist Antibodies

Polyclonal or monoclonal therapeutic anti-VEGFR-2 antibodies useful inpracticing this invention may be prepared in laboratory animals or byrecombinant DNA techniques using the following methods. Polyclonalantibodies to the VEGFR-2 molecule or a fragment thereof containing thetarget amino acid sequence generally are raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the VEGFR-2molecule in combination with an adjuvant such as Freund's adjuvant(complete or incomplete). To enhance immunogenicity, it may be useful tofirst conjugate the VEGFR-2 molecule or a fragment containing the targetamino acid sequence of a protein that is immunogenic in the species tobe immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide

(through lysine residues), glutaraldehyde, succinic anhydride, SOCl, orR¹N═C═NR, where R and R¹ are different alkyl groups. Alternatively,VEGF-2-immunogenic conjugates can be produced recombinantly as fusionproteins.

Animals are immunized against the immunogenic VEGFR-2 conjugates orderivatives (such as a fragment containing the target amino acidsequence) by combining about 1 mg or about 1 microgram of conjugate (forrabbits or mice, respectively) with about 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites.Approximately 7 to 14 days later, animals are bled and the serum isassayed for anti-VEGFR-2 titer. Animals are boosted with antigenrepeatedly until the titer plateaus. Preferably, the animal is boostedwith the same VEGFR-2 molecule or fragment thereof as was used for theinitial immunization, but conjugated to a different protein and/orthrough a different cross-linking agent. In addition, aggregating agentssuch as alum are used in the injections to enhance the immune response.

Monoclonal antibodies may be prepared by recovering spleen cells fromimmunized animals and immortalizing the cells in conventional fashion,e.g. by fusion with myeloma cells. The clones are then screened forthose expressing the desired antibody. The monoclonal antibodypreferably does not cross-react with other VEGFR family members.

Preparation of Antibodies Using Recombinant DNA Methods Such as thephagemid display method, may be accomplished using commerciallyavailable kits, as for example, the Recombinant Phagemid Antibody Systemavailable from Pharmacia (Uppsala, Sweden), or the SurfZAP™ phagedisplay system (Stratagene Inc., La Jolla, Calif.).

One may increase the population of anti-VEGFR-2 antibodies thatselectively block VEGF-A binding by using a Ig-domain 3 or otherfragment as the immunogen, but that is not necessary. After antibodiesare generated, they may be tested to ascertain their specificaffinities. Competiton studies may be performed that show that theantibody competes for binding to VEGFR-2 with VEGF-A, but not withVEGF-C.

One method comprises incubating VEGFR-2 expressing cells with eitherlabeled-VEGF-A alone, the antibody being tested alone, or with both theVEGF-A and the antibody. A label on the antibody may be employed inaddition to that on VEGF-A or instead of that label. The antibody mayalso be detected using a labeled secondary antibody. The first twogroups acting as controls allow one to confirm that both the antibodyand the VEGF-A ligand (or optionally VEGF-E) are able to bind to thereceptor in the absence of the other. Those cell samples treated withboth VEGF-A (or VEGF-E) and an antibody, that reveal binding of theantibody, but not VEGF-A (or VEGF-E) indicate that the antibody shouldbe further tested. As described below, stoichiometric analysis can beused to ascertain that the ligand and antibody are competing for thesame molecule.

This further testing may comprise binding studies that reveal that bothVEGF-C (or VEGF-D) and the antibody are able to bind the receptorsimultaneously. This testing also is designed to determine whetherVEGF-C and the antibody are simultaneously binding to a single VEGFR-2molecule as opposed to binding of VEGF-C and the antibody binding todifferent VEGFR-2 molecules. Comparative quantitative binding studiesmay accordingly be used. The VEGFR-2 cells are counted in each sample.VEGFR-2 samples, having been counted, are incubated with either labeledVEGF-C alone or labeled (or unlabled using a secondary antibody fordetection) antibody alone. The degree of binding is measured,quantitated, using suitable imaging procedures, e.g., if radiolabel isemployed using a phosphoimager. The average number of VEGFR-2 receptorsper cell are calculated by dividing the amount of bound molecules by thetotal number of cells. Whether the receptors are saturated withmolecules may be achieved by repeating the assay with increasing amountsof the labeled molecule(s). The binding assay is repeated again withboth ligand and antibody. If the quantification reveals that the numberof antibodies and ligands bound is greater than the total number ofreceptors, then the antibody has the desired characteristics.

The described protocols may also be modified and used to produceantibodies against any of the other antigens identified herein astargets for anti-rejection therapy, including but not limited toVEGFR-3, VEGF-C, VEGF-D, the other VEGF growth factors, the PDGFreceptors, and the PDGF growth factors.

Preferably, antibodies for administration to humans, although preparedin a laboratory animal such as a mouse, will be “humanized”, orchimeric, i.e. made to be compatible with the human immune system suchthat a human patient will not develop an immune response to theantibody. Even more preferably, human antibodies which can now beprepared using methods such as those described for example, in Lonberg,et al., Nature Genetics, 7:13-21 (1994) are preferred for therapeuticadministration to patients. Fully human antibodies are highly preferred.

1. Humanization Of Anti-VEGFR-2 Monoclonal Antibodies

Selective binding agents, including monoclonal antibodies, whichselectively block VEGF-A without blocking VEGF-C (or VEGF-D) binding maybe applied therapeutically. Following are protocols to improve theutility of anti-VEGFR-2 monoclonal antibodies as therapeutics in humans,by “humanizing” the monoclonal antibodies to improve their serumhalf-life and render them less immunogenic in human hosts (i.e., toprevent human antibody response to non-human anti-VEGFR-2 antibodies).The description also applies to antibodies directed to the otherantigens described herein.

The principles of humanization have been described in the literature andare facilitated by the modular arrangement of antibody proteins. Tominimize the possibility of binding complement, a humanized antibody ofthe IgG4 isotype is preferred.

For example, a level of humanization is achieved by generating chimericantibodies comprising the variable domains of non-human antibodyproteins of interest, such as the anti-VEGFR-2 monoclonal antibodiesdescribed herein, with the constant domains of human antibody molecules.(See, e.g., Morrison and Oi, Adv. Immunol., 44:65-92 (1989).) Thevariable domains of VEGFR-2 neutralizing anti-VEGFR-2 antibodies arecloned from the genomic DNA of a B-cell hybridoma or from cDNA generatedfrom mRNA isolated from the hybridoma of interest. The V region genefragments are linked to exons encoding human antibody constant domains,and the resultant construct is expressed in suitable mammalian hostcells (e.g., myeloma or CHO cells).

To achieve an even greater levels of humanization, only those portionsof the variable region gene fragments that encode antigen-bindingcomplementarity determining regions (“CDR”) of the non-human monoclonalantibody genes are cloned into human antibody sequences. [See, e.g.,Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-36 (1988); andTempest et al., Bio/Technology, 9:266-71 (1991).] If necessary, theB-sheet framework of the human antibody surrounding the CDR3 regionsalso is modified to more closely mirror the three dimensional structureof the antigen-binding domain of the original monoclonal antibody. [(SeeKettleborough et al., Protein Engin., 4:773-783 (1991); and Foote etal., J. Mol. Biol., 224:487-499 (1992).)]

In an alternative approach, the surface of a non-human monoclonalantibody of interest is humanized by altering selected surface residuesof the non-human antibody, e.g., by site-directed mutagenesis, whileretaining all of the interior and contacting residues of the non-humanantibody. [See Padlan, Molecular Immunol., 28(4/5):489-98 (1991).]

The foregoing approaches are employed using VEGFR-2-neutralizinganti-VEGFR-2 monoclonal antibodies and the hybridomas that produce themto generate humanized VEGFR-2-neutralizing antibodies useful astherapeutics to treat or palliate conditions wherein VEGFR-2 expressionis detrimental and/or activation by VEGF-A. One therapeutic target isselective promotion of lymphangiogenesis while minimizing promotion ofangiogenesis.

2. Human VEGFR-2-Neutralizing Antibodies From Phage Display

Human VEGFR-2-neutralizing antibodies are generated by phage displaytechniques such as those described in Aujame et al., Human Antibodies,8(4):155-168 (1997); Hoogenboom, TIBTECH, 15:62-70 (1997); and Rader etal., Curr. Opin. Biotechnol., 8:503-508 (1997), all of which areincorporated by reference. For example, antibody variable regions in theform of Fab fragments or linked single chain Fv fragments are fused tothe amino terminus of filamentous phage minor coat protein pIII.Expression of the fusion protein and incorporation thereof into themature phage coat results in phage particles that present an antibody ontheir surface and contain the genetic material encoding the antibody. Aphage library comprising such constructs is expressed in bacteria, andthe library is panned (screened) for VEGFR-2-specific phage-antibodiesusing labeled or immobilized VEGFR-2 as antigen-probe.

3. Human VEGFR-2-Neutralizing Antibodies From Transgenic Mice

Human VEGFR-2-neutralizing antibodies are generated in transgenic miceessentially as described in Bruggemann and Neuberger, Immunol. Today,17(8):391-97 (1996) and Bruggemann and Taussig, Curr. Opin. Biotechnol.,8:455-58 (1997). Transgenic mice carrying human V-gene segments ingermline configuration and that express these transgenes in theirlymphoid tissue are immunized with an VEGFR-2 composition usingconventional immunization protocols. Hybridomas are generated using Bcells from the immunized mice using conventional protocols and screenedto identify hybridomas secreting anti-VEGFR-2 human antibodies (e.g., asdescribed above).

4. Bispecific Antibodies

Bispecific antibodies that specifically bind to VEGFR-2 and thatspecifically bind to other antigens relevant to pathology and/ortreatment are produced, isolated, and tested using standard proceduresthat have been described in the literature. See, e.g., Pluckthun & Pack,Immunotechnology, 3:83-105 (1997); Carter et al., J. Hematotherapy, 4:463-470 (1995); Renner & Pfreundschuh, Immunological Reviews, 1995, No.145, pp. 179-209; Pfreundschuh U.S. Pat. No. 5,643,759; Segal et al., J.Hematotherapy, 4: 377-382 (1995); Segal et al., Immunobiology, 185:390-402 (1992); and Bolhuis et al., Cancer Immunol. Immunother., 34: 1-8(1991), all of which are incorporated herein by reference in theirentireties. Bispecific antibodies that may be employed in combinationwith the binding constructs of the invention include those described inU.S. Patent Publication No. 2005/0282233, incorporated herein byreference.

For example, bispecific antibodies (bscAb) are produced by joining twosingle-chain Fv fragments via a glycine-serine linker using recombinantmethods. The V light-chain (V_(L)) and V heavy-chain (V_(H)) domains oftwo antibodies of interest are isolated using standard PCR methods. TheV_(L) and V_(H) cDNA's obtained from each hybridoma are then joined toform a single-chain fragment in a two-step fusion PCR. Bispecific fusionproteins are prepared in a similar manner. Bispecific single-chainantibodies and bispecific fusion proteins are antibody substancesincluded within the scope of the present invention.

Antibody fragments that contain the antigen binding, or idiotype, of themolecule may be generated by known techniques. For example, suchfragments include, but are not limited to, the F(ab′)₂ fragment whichmay be produced by pepsin digestion of the antibody molecule; the Fab′fragments which may be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragment, and the two Fab′ fragments which may be generatedby treating the antibody molecule with papain and a reducing agent.

Chemically constructed bispecific antibodies may be prepared bychemically cross-linking heterologous Fab or F(ab′)₂ fragments by meansof chemicals such as heterobifunctional reagentsuccinimidyl-3-(2-pyridyldithiol)-propionate (SPDP, Pierce Chemicals,Rockford, Ill.). The Fab and F(ab′)₂ fragments can be obtained fromintact antibody by digesting it with papain or pepsin, respectively(Karpovsky et al., J. Exp. Med. 160:1686-701, 1984; Titus et al., J.Immunol., 138:4018-22, 1987).

5. Humanization of Known Anti-VEGFR-2 Antibodies

Existing anti-VEGF-2 antibodies may also be employed in the variousmethods and compositions of the present invention, and, if not alreadyhumanized, may be humanized as discussed herein. Known anti-VEGFR-2antibodies may be tested for the ability to selectively block VEGF-Abinding using the methods discussed herein. Known anti-VEGFR-2antibodies (anti-KDR antibodies) are taught for example in Lu et al., J.Immunological Methods, 230:159-71 (1999); Lu, et al., J. Biol. Chem.,275(19): 14321-14330 (2000); and Lu, et al., J. Biol. Chem., 278(44):43496-43507 (2003).

6. Domain Antibodies

A domain antibody comprises a functional binding unit of an antibody,and can correspond to the variable regions of either the heavy (V_(H))or light (V_(L)) chains of antibodies. A domain antibody can have amolecular weight of approximately 13 kDa, or approximately one-tenth ofa full antibody. Domain antibodies may be derived from full antibodiessuch as those described herein.

B. Anti-Receptor And Anti-Ligand Aptamers

Recent advances in the field of combinatorial sciences have identifiedshort polymer sequences with high affinity and specificity to a giventarget. For example, SELEX technology has been used to identify DNA andRNA aptamers with binding properties that rival mammalian antibodies,the field of immunology has generated and isolated antibodies orantibody fragments which bind to a myriad of compounds and phage displayhas been utilized to discover new peptide sequences with very favorablebinding properties. Based on the success of these molecular evolutiontechniques, it is certain that molecules can be created which bind toany target molecule. A loop structure is often involved with providingthe desired binding attributes as in the case of: aptamers which oftenutilize hairpin loops created from short regions without complimentarybase pairing, naturally derived antibodies that utilize combinatorialarrangement of looped hyper-variable regions and new phage displaylibraries utilizing cyclic peptides that have shown improved resultswhen compared to linear peptide phage display results. Thus, sufficientevidence has been generated to suggest that high affinity ligands can becreated and identified by combinatorial molecular evolution techniques.For the present invention, molecular evolution techniques can be used toisolate binding constructs specific for ligands described herein. Formore on aptamers, See generally, Gold, L., Singer, B., He, Y. Y., Brody.E., “Aptamers As Therapeutic And Diagnostic Agents,” J. Biotechnol.74:5-13 (2000). Relevant techniques for generating aptamers may be foundin U.S. Pat. No. 6,699,843, which is incorporated by reference in itsentirety.

In some embodiments, the aptamer may be generated by preparing a libraryof nucleic acids; contacting the library of nucleic acids with a growthfactor, wherein nucleic acids having greater binding affinity for thegrowth factor (relative to other library nucleic acids) are selected andamplified to yield a mixture of nucleic acids enriched for nucleic acidswith relatively higher affinity and specificity for binding to thegrowth factor. The processes may be repeated, and the selected nucleicacids mutated and rescreened, whereby a growth factor aptamer is beidentified. Nucleic acids may be screened to select for molecules thatbind to more than growth factor. Binding more than one growth factor canrefer to binding more than one growth factor simultaneously orcompetitively. In some embodiments a binding construct will comprise atleast one aptamer, wherein a first binding unit binds VEGF-A and asecond binding unit binds VEGF-C. In some embodiments a bindingconstruct will comprise at least one aptamer, wherein a first bindingunit binds a VEGF growth factor subfamily member and a second bindingunit binds a PDGF subfamily member.

C. Anti-Sense Molecules And Therapy

Another class of inhibitors that may be used in conjunction with thepresent invention is isolated antisense nucleic acid molecules that canhybridize to, or are complementary to, the nucleic acid molecule,nucleotide sequence, or fragments, analogs or derivatives thereof.Antisense modulation of VEGF-C is described in U.S. Patent ApplicationPublication No. 2003/0232437, the disclosure of which is incorporatedherein by reference in its entirety. Antisense modulation of VEGFR-2 isdescribed in U.S. Pat. No. 6,734,017, the disclosure of which isincorporated herein by reference in its entirety.

Antisense and interfering RNA molecules that target any of the growthfactors (e.g., VEGF-A, -B, -C, -D; PDGF-A, -B, -C, -D) and growth factorreceptors (e.g., VEGFR-1, VEGFR-2, VEGFR-3, PDGFR-alpha, PDGFR-beta)described herein are specifically contemplated for use in methods andproducts of the invention.

An “antisense” nucleic acid comprises a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding a protein (e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence). In specific embodiments, antisensenucleic acid molecules are provided that comprise a sequencecomplementary to at least about 10, 25, 50, 100, 250 or 500 nucleotidesor an entire receptor or ligand coding strand, or to only a portionthereof. Nucleic acid molecules encoding fragments, homologs,derivatives and analogs of receptor or ligand or antisense nucleic acidscomplementary to a receptor or ligand nucleic acid sequence areadditionally provided.

In one embodiment, an antisense nucleic acid molecule is antisense to a“coding region” of the coding strand of a nucleotide sequence encoding areceptor or ligand protein (or fragments or fragment combinationthereof). The term “coding region” refers to the region of thenucleotide sequence comprising codons that are translated into aminoacid residues. In another embodiment, the antisense nucleic acidmolecule is antisense to a “conceding region” of the coding strand of anucleotide sequence encoding the receptor or ligand protein. The term“conceding region” refers to 5′ and 3′ sequences that flank the codingregion and that are not translated into amino acids (i.e., also referredto as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding the receptor or ligandprotein disclosed herein, antisense nucleic acids of the invention canbe designed according to the rules of Watson and Crick or Hoogsteen basepairing. The antisense nucleic acid molecule can be complementary to theentire coding region of a ligand or receptor mRNA, but more preferablyis an oligonucleotide that is antisense to only a portion of the codingor noncoding region of receptor or ligand mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of receptor or ligand mRNA. An antisenseoligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35,40, 45, or 50 nucleotides in length. An antisense nucleic acid of theinvention can be constructed using chemical synthesis or enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally-occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids (e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused).

Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following section).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a receptor orligand to thereby inhibit expression of the protein (e.g., by inhibitingtranscription and/or translation). The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule that binds toDNA duplexes, through specific interactions in the major groove of thedouble helix. An example of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface (e.g., by linking the antisensenucleic acid molecules to peptides or antibodies that bind to cellsurface receptors or antigens). The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient nucleic acid molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an alpha-anomeric nucleic acid molecule. An alpha-anomericnucleic acid molecule forms specific double-stranded hybrids withcomplementary RNA in which, contrary to the usual alpha-units, thestrands run parallel to each other. See, e.g., Gaultier, et al., Nucl.Acids Res., 15:6625-6641 (1987). The antisense nucleic acid molecule canalso comprise a 2′-o-methylribonucleotide (see, e.g., Inoue, et al.Nucl. Acids Res., 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue(see, e.g., Inoue, et al., FEBS Lett., 215:327-330 (1987)).

Production and delivery of antisense molecules are facilitated byproviding a vector comprising an anti-sense nucleotide sequencecomplementary to at least a part of the Receptor or ligand DNA sequence.According to a yet further aspect of the invention such a vectorcomprising an anti-sense sequence may be used to inhibit, or at leastmitigate, Receptor or ligand expression.

Alternatively, nucleic acid sequences which inhibit or interfere withgene expression (e.g., siRNA, shRNA, ribozymes, aptamers) can be used toinhibit or interfere with the activity of RNA or DNA encoding a targetprotein.

D. Anti-Ligand or Anti-Receptor RNA Interference

Use of RNA Interference to inactivate or modulate receptor or ligandexpression is also contemplated by this invention. RNA interference isdescribed in U.S. Patent Appl. Pub. No. 2002/0162126, and Hannon, G., J.Nature, 11:418:244-51 (2002). “RNA interference,” “post-transcriptionalgene silencing,” “quelling”—these terms have all been used to describesimilar effects that result from the overexpression or misexpression oftransgenes, or from the deliberate introduction of double-stranded RNAinto cells (reviewed in Fire, A., Trends Genet. 15:358-363 (1999);Sharp, P. A., Genes Dev., 13:139-141 (1999); Hunter, C., Curr. Biol.,9:R440-R442 (1999); Baulcombe, D. C., Curr. Biol. 9:R599-R601 (1999);Vaucheret, et al. Plant J. 16:651-659 (1998), all incorporated byreference. RNA interference, commonly referred to as RNAi, offers a wayof specifically and potently inactivating a cloned gene. RNAinterference of the VEGF family of proteins and receptors is describedin U.S. Patent application Publication Nos.: 2006/0217332, 2006/0025370,2005/0233998, 2005/0222066 and 2005/0171039, the disclosure of which areincorporated herein by reference in their entireties.

Interfering RNA directed to VEGF or VEGFR family members is described inU.S. Patent Publication No. 2006/0217332, incorporated herein byreference.

siRNA (short interfering RNA) technology relates to a process ofsequence-specific post-transcriptional gene repression which can occurin eukaryotic cells. In general, this process involves degradation of anmRNA of a particular sequence induced by double-stranded RNA (dsRNA)that is homologous to that sequence. For example, the expression of along dsRNA corresponding to the sequence of a particular single-strandedmRNA (ss mRNA) will labilize that message, thereby “interfering” withexpression of the corresponding gene. Accordingly, any selected gene maybe repressed by introducing a dsRNA which corresponds to all or asubstantial part of the mRNA for that gene. It appears that when a longdsRNA is expressed, it is initially processed by a ribonuclease III intoshorter dsRNA oligonucleotides of as few as 21 to 22 base pairs inlength. Accordingly, siRNA may be effected by introduction or expressionof relatively short homologous dsRNAs. Indeed the use of relativelyshort homologous dsRNAs may have certain advantages as discussed below.

Compared to siRNA, shRNA (short hairpin RNA) offers advantages insilencing longevity and delivery options. See, Hannon et al., Nature,431:371-378, 2004, for review. Vectors that produce shRNAs, which areprocessed intracellularly into short duplex RNAs having siRNA-likeproperties have been reported (Brummelkamp et al., Science 296, 550-553,2000; Paddison et al., Genes Dev. 16, 948-958 (2002). Such vectorsprovide a renewable source of a gene-silencing reagent that can mediatepersistent gene silencing after stable integration of the vector intothe host-cell genome. Furthermore, the core silencing ‘hairpin’ cassettecan be readily inserted into retroviral, lentiviral or adenoviralvectors, facilitating delivery of shRNAs into a broad range of celltypes (Brummelkamp et al., Cancer Cell 2:243-247, 2002; Dirac, et al.,J. Biol. Chem. 278:11731-11734, 2003; Michiels et al., Nat. Biotechnol.20:1154-1157, 2002; Stegmeie et al., Proc. Natl. Acad. Sci. USA102:13212-13217, 2005; Khvorova et al., Cell, 115:209-216 (2003) in anyof the innumerable ways that have been devised for delivery of DNAconstructs that allow ectopic mRNA expression. These include standardtransient transfection, stable transfection and delivery using virusesranging from retroviruses to adenoviruses. Expression can also be drivenby either constitutive or inducible promoter systems (Paddison et al.,Methods Mol. Biol. 265:85-100, 2004). Delivery of nucleic acidinhibitors by replicating or replication-deficient vectors iscontemplated as an aspect of the invention.

Mammalian cells have at least two pathways that are affected bydouble-stranded RNA (dsRNA). In the siRNA (sequence-specific) pathway,the initiating dsRNA is first broken into short interfering (si) RNAs,as described above. The siRNAs have sense and antisense strands of about21 nucleotides that form approximately 19 nucleotide si RNAs withoverhangs of two nucleotides at each 3′ end. Short interfering RNAs arethought to provide the sequence information that allows a specificmessenger RNA to be targeted for degradation. In contrast, thenonspecific pathway is triggered by dsRNA of any sequence, as long as itis at least about 30 base pairs in length.

The nonspecific effects occur because dsRNA activates two enzymes: PKR,which in its active form phosphorylates the translation initiationfactor eIF2 to shut down all protein synthesis, and 2′, 5′oligoadenylate synthetase (2′,5′-AS), which synthesizes a molecule thatactivates RNase L, a nonspecific enzyme that targets all mRNAs. Thenonspecific pathway may represent a host response to stress or viralinfection, and, in general, the effects of the nonspecific pathway arepreferably minimized. Significantly, longer dsRNAs appear to be requiredto induce the nonspecific pathway and, accordingly, dsRNAs shorter thanabout 30 bases pairs are preferred to effect gene repression by RNAi(see Hunter et al., 1975, J. Biol. Chem. 250:409-17; Manche et al.,1992, Mol. Cell. Biol. 12:5239-48; Minks et al., 1979, J. Biol. Chem.254:10180-3; and Elbashir et al., 2001, Nature 411:494-8). siRNA hasproven to be an effective means of decreasing gene expression in avariety of cell types including HeLa cells, NIH/3T3 cells, COS cells,293 cells and BHK-21 cells, and typically decreases expression of a geneto lower levels than that achieved using antisense techniques and,indeed, frequently eliminates expression entirely (see Bass, 2001,Nature 411:428-9). In mammalian cells, siRNAs are effective atconcentrations that are several orders of magnitude below theconcentrations typically used in antisense experiments (Elbashir et al.,2001, Nature 411:494-8).

The double stranded oligonucleotides used to effect RNAi are preferablyless than 30 base pairs in length and, more preferably, comprise about25, 24, 23, 22, 21, 20, 19, 18 or 17 base pairs of ribonucleic acid.Optionally the dsRNA oligonucleotides may include 3′ overhang ends.Exemplary 2-nucleotide 3′ overhangs may be composed of ribonucleotideresidues of any type and may even be composed of 2′-deoxythymidineresides, which lowers the cost of RNA synthesis and may enhance nucleaseresistance of siRNAs in the cell culture medium and within transfectedcells (see Elbashi et al., 2001, Nature 411:494-8).

[Longer dsRNAs of 50, 75, 100 or even 500 base pairs or more may also beutilized in certain embodiments of the invention. Exemplaryconcentrations of dsRNAs for effecting RNAi are about 0.05 nM, 0.1 nM,0.5 nM, 1.0 nM, 1.5 nM, 25 nM or 100 nM, although other concentrationsmay be utilized depending upon the nature of the cells treated, the genetarget and other factors readily discernable to the skilled artisan.

Exemplary dsRNAs may be synthesized chemically or produced in vitro orin vivo using appropriate expression vectors. Exemplary synthetic RNAsinclude 21 nucleotide RNAs chemically synthesized using methods known inthe art. Synthetic oligonucleotides are preferably deprotected andgel-purified using methods known in the art (see e.g. Elbashir et al.,2001, Genes Dev. 15:188-200). Longer RNAs may be transcribed frompromoters, such as T7 RNA polymerase promoters, known in the art. Asingle RNA target, placed in both possible orientations downstream of anin vitro promoter, will transcribe both strands of the target to createa dsRNA oligonucleotide of the desired target sequence. Any of the aboveRNA species will be designed to include a portion of nucleic acidsequence represented in a target nucleic acid.

The specific sequence utilized in design of the oligonucleotides may beany contiguous sequence of nucleotides contained within the expressedgene message of the target. Programs and algorithms, known in the art,may be used to select appropriate target sequences. In addition, optimalsequences may be selected utilizing programs designed to predict thesecondary structure of a specified single stranded nucleic acid sequenceand allowing selection of those sequences likely to occur in exposedsingle stranded regions of a folded mRNA. Methods and compositions fordesigning appropriate oligonucleotides may be found, for example, inU.S. Pat. No. 6,251,588, the contents of which are incorporated hereinby reference.

Although mRNAs are generally thought of as linear molecules containingthe information for directing protein synthesis within the sequence ofribonucleotides, most mRNAs have been shown to contain a number ofsecondary and tertiary structures. Secondary structural elements in RNAare formed largely by Watson-Crick type interactions between differentregions of the same RNA molecule. Important secondary structuralelements include intramolecular double stranded regions, hairpin loops,bulges in duplex RNA and internal loops. Tertiary structural elementsare formed when secondary structural elements come in contact with eachother or with single stranded regions to produce a more complex threedimensional structure. A number of researchers have measured the bindingenergies of a large number of RNA duplex structures and have derived aset of rules which can be used to predict the secondary structure of RNA(see e.g. Jaeger et al., 1989, Proc. Natl. Acad. Sci. USA 86:7706; andTurner et al., 1988, Annu. Rev. Biophys. Biophys. Chem. 17:167). Therules are useful in identification of RNA structural elements and, inparticular, for identifying single stranded RNA regions which mayrepresent preferred segments of the mRNA to target for siRNA, ribozymeor antisense technologies. Accordingly, preferred segments of the mRNAtarget can be identified for design of the siRNA mediating dsRNAoligonucleotides as well as for design of appropriate ribozyme andhammerheadribozyme compositions of the invention (see below).

The dsRNA oligonucleotides may be introduced into the cell bytransfection with a heterologous target gene using carrier compositionssuch as liposomes, which are known in the art—e.g. Lipofectamine 2000(Life Technologies) as described by the manufacturer for adherent celllines. Transfection of dsRNA oligonucleotides for targeting endogenousgenes may be carried out using Oligofectamine (Life Technologies).Transfection efficiency may be checked using fluorescence microscopy formammalian cell lines after co-transfection of hGFP-encoding pAD3(Kehlenback et al., 1998, J. Cell Biol. 141:863-74). The effectivenessof the siRNA may be assessed by any of a number of assays followingintroduction of the dsRNAs. These include Western blot analysis usingantibodies which recognize the target gene product following sufficienttime for turnover of the endogenous pool after new protein synthesis isrepressed, reverse transcriptase polymerase chain reaction and Northernblot analysis to determine the level of existing target mRNA.

Further compositions, methods and applications of siRNA technology areprovided in U.S. patent application Nos. 6,278,039, 5,723,750 and5,244,805, which are incorporated herein by reference.

E. Small Molecule Inhibitors

Any chemical substance that can be safely administered as a therapeuticand that can be used to modulate biochemical pathway targets identifiedherein, such as VEGF-mediated stimulation of VEGF receptors, may be usedto practice the invention. Small molecules that inhibit the interactionbetween VEGF-C and/or VEGFR-3 with VEGFR-3 are specificallycontemplated. VEGF-C/VEGF-D inhibitors are disclosed in U.S. Pat. No.7,045,133, incorporated herein by reference.

The VEGF receptors are receptor tyrosine kinases and intracellularsignaling is initiated through receptor phosphorylation. Accordingly,one preferred class of molecules for practice of the invention istyrosine kinase inhibitors, including those described in and Morin,Oncogene, 19(56):6574-83, 2000, incorporated herein by reference.VEGFR-3 inhibitors are disclosed in U.S. Patent Publication No.2002-0164667, incorporated herein by reference.

IV. THERAPEUTIC FORMULATIONS AND ADMINISTRATION

A. Therapeutic Formulations

Binding constructs, or polynucleotides encoding the same, can be useddirectly to practice materials and methods of the invention, but inpreferred embodiments, the compounds are formulated withpharmaceutically acceptable diluents, adjuvants, excipients, orcarriers. The phrase “pharmaceutically or pharmacologically acceptable”refers to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human, e.g., orally, topically, transdermally, parenterally,by inhalation spray, vaginally, rectally, or by intracranial injection.(The term parenteral as used herein includes subcutaneous injections,intravenous, intramuscular, intracisternal injection, or infusiontechniques. Administration by intravenous, intradermal, intramusclar,intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonaryinjection and/or surgical implantation at a particular site iscontemplated as well.) Generally, this will also entail preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals. The term“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art.

Therapeutic formulations of the compositions useful for practicing theinvention such as polypeptides, polynucleotides, or antibodies may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional physiologicallypharmaceutically-acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, ed.,Mack Publishing Company (1990)) in the form of a lyophilized cake or anaqueous solution. Pharmaceutical compositions may be produced byadmixing with one or more suitable carriers or adjuvants such as water,mineral oil, polyethylene glycol, starch, talcum, lactose, thickeners,stabilizers, suspending agents, etc. Such compositions may be in theform of solutions, suspensions, tablets, capsules, creams, salves,ointments, or other conventional forms.

Acceptable carriers, excipients or stabilizers are nontoxic torecipients and are preferably inert at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, or otherorganic acids; antioxidants such as ascorbic acid; low molecular weightpolypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics orpolyethylene glycol (PEG).

The composition to be used for in vivo administration should be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle. The routeof administration of the composition is in accord with known methods,e.g. oral, injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, orintralesional routes, or by sustained release systems or implantationdevice. Where desired, the compositions may be administered continuouslyby infusion, bolus injection or by implantation device. The compositionfor parenteral administration ordinarily will be stored in lyophilizedform or in solution.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form should be sterile and should be fluidto the extent that easy syringability exists. It should be stable underthe conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Suitable examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman, et al.,Biopolymers, 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate)(Langer, et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer,Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer, et al.,supra) or poly-D(−)-3-hydroxybutyric acid (EP 133,988).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (e.g., DE 3,218,121;Epstein, et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang,et al., Proc. Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949).

An effective amount of the compositions to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. A therapist can titerthe dosage and modify the route of administration to obtain the optimaltherapeutic effect. A typical daily dosage may range from about 1 μg/kgto up to 100 mg/kg or more, depending on the factors mentioned above.Typically, a clinician will administer the composition until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays designed to evaluate theparticular disease state being treated.

B. Kits And Unit Doses

In related variations of the preceding embodiments, a binding constructmay be packaged or formulated together with another binding construct orother therapeutic (e.g., an immunosuppressive agent), e.g., in a kit orpackage or unit dose, to permit co-administration, but these twocomponents are not in admixture. In some embodiments, the two componentsto the kit/unit dose are packaged with instructions for administeringthe two compounds to a human subject for treatment of one of thedisorders and diseases described herein.

C. Polynucleotide-Based Therapies

The present invention also includes gene therapy materials and methods.Specifically, polypeptides and binding constructions of the inventioncan be produced at therapeutic levels in vivo by administration of agene therapy contrast that enters cells and is expressed in vivo toproduce the polypeptides or binding constructs. For example, in someembodiments, the vasculature of a cancer cell or cancer cells may becontacted with an expression construct capable of providing atherapeutic peptide or binding constructs of the present invention.Expression of the polypeptide or binding construct causes a therapeuticoutcome, for example, inhibition of growth factors and receptors in thevasculature of a tumor, an inhibition of angiogenesis, an inhibition oflymphangiogenesis, an ablation, regression or other inhibition of tumorgrowth, an induction of apoptosis of the blood or lymphatic vasculatureof the tumor or indeed the tumor cells themselves.

For these embodiments, an exemplary expression construct comprises avirus or engineered construct derived from a viral genome. Such vectorsand constructs are considered aspect of the invention. The expressionconstruct generally comprises a nucleic acid encoding the gene orbinding construct, including any nucleic acid molecule described herein,to be expressed and also additional regulatory regions that will effectthe expression of the gene in the cell to which it is administered. Suchregulatory regions include for example promoters, enhancers,polyadenylation signals and the like.

DNA may be introduced into a cell using a variety of viral vectors. Insuch embodiments, expression constructs comprising viral vectorscontaining the genes of interest may be adenoviral (see, for example,U.S. Pat. No. 5,824,544; U.S. Pat. No. 5,707,618; U.S. Pat. No.5,693,509; U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,585,362, eachincorporated herein by reference), retroviral (see, for example, U.S.Pat. No. 5,888,502; U.S. Pat. No. 5,830,725; U.S. Pat. No. 5,770,414;U.S. Pat. No. 5,686,278; U.S. Pat. No. 4,861,719, each incorporatedherein by reference), adeno-associated viral (see, for example, U.S.Pat. No. 5,474,935; U.S. Pat. No. 5,139,941; U.S. Pat. No. 5,622,856;U.S. Pat. No. 5,658,776; U.S. Pat. No. 5,773,289; U.S. Pat. No.5,789,390; U.S. Pat. No. 5,834,441; U.S. Pat. No. 5,863,541; U.S. Pat.No. 5,851,521; U.S. Pat. No. 5,252,479, each incorporated herein byreference), an adenoviral-adenoassociated viral hybrid (see, forexample, U.S. Pat. No. 5,856,152 incorporated herein by reference) or avaccinia viral or a herpesviral (see, for example, U.S. Pat. No.5,879,934; U.S. Pat. No. 5,849,571; U.S. Pat. No. 5,830,727; U.S. Pat.No. 5,661,033; U.S. Pat. No. 5,328,688, each incorporated herein byreference) vector. Other vectors described herein may also be employed.Replication-deficient viral vectors are specifically contemplated.

In other embodiments, non-viral delivery is contemplated. These includecalcium phosphate precipitation (Graham and Van Der Eb, Virology,52:456-467 (1973); Chen and Okayama, Mol. Cell. Biol., 7:2745-2752,(1987); Rippe, et al., Mol. Cell. Biol., 10:689-695 (1990)),DEAE-dextran (Gopal, Mol. Cell. Biol., 5:1188-1190 (1985)),electroporation (Tur-Kaspa, et al., Mol. Cell. Biol., 6:716-718, (1986);Potter, et al., Proc. Nat. Acad. Sci. USA, 81:7161-7165, (1984)), directmicroinjection (Harland and Weintraub, J. Cell Biol., 101:1094-1099(1985)), DNA-loaded liposomes (Nicolau and Sene, Biochim. Biophys. Acta,721:185-190 (1982); Fraley, et al., Proc. Natl. Acad. Sci. USA,76:3348-3352 (1979); Felgner, Sci. Am., 276(6):102-6 (1997); Felgner,Hum. Gene Ther., 7(15):1791-3, (1996)), cell sonication (Fechheimer, etal., Proc. Natl. Acad. Sci. USA, 84:8463-8467 (1987)), gene bombardmentusing high velocity microprojectiles (Yang, et al., Proc. Natl. Acad.Sci. USA, 87:9568-9572 (1990)), and receptor-mediated transfection (Wuand Wu, J. Biol. Chem., 262:4429-4432 (1987); Wu and Wu, Biochemistry,27:887-892 (1988); Wu and Wu, Adv. Drug Delivery Rev., 12:159-167(1993)).

In a particular embodiment of the invention, the expression construct(or indeed the peptides discussed above) may be entrapped in a liposome.Liposomes are vesicular structures characterized by a phospholipidbilayer membrane and an inner aqueous medium. Multilamellar liposomeshave multiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers (Ghosh and Bachhawat, “In Liver Diseases,Targeted Diagnosis And Therapy Using Specific Receptors And Ligands,”Wu, G., Wu, C., ed., New York: Marcel Dekker, pp. 87-104 (1991)). Theaddition of DNA to cationic liposomes causes a topological transitionfrom liposomes to optically birefringent liquid-crystalline condensedglobules (Radler, et al., Science, 275(5301):810-4, (1997)). TheseDNA-lipid complexes are potential non-viral vectors for use in genetherapy and delivery.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Also contemplated in the presentinvention are various commercial approaches involving “lipofection”technology. In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda, et al., Science, 243:375-378 (1989)).In other embodiments, the liposome may be complexed or employed inconjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato,et al., J. Biol. Chem., 266:3361-3364 (1991)). In yet furtherembodiments, the liposome may be complexed or employed in conjunctionwith both HVJ and HMG-1. In that such expression constructs have beensuccessfully employed in transfer and expression of nucleic acid invitro and in vivo, then they are applicable for the present invention.

Other vector delivery systems that can be employed to deliver a nucleicacid encoding a therapeutic gene into cells include receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu (1993),supra).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu (1987), supra) and transferrin (Wagner, et al., Proc. Nat'l.Acad. Sci. USA, 87(9):3410-3414 (1990)). Recently, a syntheticneoglycoprotein, which recognizes the same receptor as ASOR, has beenused as a gene delivery vehicle (Ferkol, et al., FASEB. J., 7:1081-1091(1993); Perales, et al., Proc. Natl. Acad. Sci., USA 91:4086-4090(1994)) and epidermal growth factor (EGF) has also been used to delivergenes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau, et al., Methods Enzymol., 149:157-176(1987) employed lactosyl-ceramide, a galactose-terminalasialganglioside, incorporated into liposomes and observed an increasein the uptake of the insulin gene by hepatocytes. Thus, it is feasiblethat a nucleic acid encoding a therapeutic gene also may be specificallydelivered into a particular cell type by any number of receptor-ligandsystems with or without liposomes.

In another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above thatphysically or chemically permeabilize the cell membrane. This isapplicable particularly for transfer in vitro, however, it may beapplied for in vivo use as well. Dubensky, et al., Proc. Nat. Acad. Sci.USA, 81:7529-7533 (1984) successfully injected polyomavirus DNA in theform of CaPO₄ precipitates into liver and spleen of adult and newbornmice demonstrating active viral replication and acute infection.Benvenisty and Neshif, Proc. Nat. Acad. Sci. USA, 83:9551-9555 (1986)also demonstrated that direct intraperitoneal injection of CaPO₄precipitated plasmids results in expression of the transfected genes.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein, et al., Nature, 327:70-73 (1987)).Several devices for accelerating small particles have been developed.One such device relies on a high voltage discharge to generate anelectrical current, which in turn provides the motive force (Yang, etal., Proc. Natl. Acad. Sci. USA, 87:9568-9572 (1990)). Themicroprojectiles used have consisted of biologically inert substancessuch as tungsten or gold beads.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10⁴, 1×10¹, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the patient.Similar figures may be extrapolated for liposomal or other non-viralformulations by comparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below.

Various routes are contemplated for various cell types. For practicallyany cell, tissue or organ type, systemic delivery is contemplated. Inother embodiments, a variety of direct, local and regional approachesmay be taken. For example, the cell, tissue or organ may be directlyinjected with the expression vector or protein.

Promoters for gene therapy for use in this invention includecytomegalovirus (CMV) promoter/enhancer, long terminal repeat (LTR) ofretroviruses, keratin 14 promoter, and a myosin heavy chain promoter.

In a different embodiment, ex vivo gene therapy is contemplated. In anex vivo embodiment, cells from the patient are removed and maintainedoutside the body for at least some period of time. During this period, atherapy is delivered, after which the cells are reintroduced into thepatient; preferably, any tumor cells in the sample have been killed.

The techniques, procedures and methods outlined herein are applicable toany and all of the polypeptides and binding constructs of the presentinvention.

D. Immunosuppressive And Other Combination Therapies

Any one of the binding constructs of the present invention when used ina method of treating or preventing a disease, e.g, a graft rejection orarteriosclerosis, may be employed alone, or in combination with otheragents. In some embodiments, more than one binding construct may beadministered. In some embodiments, a binding construct may beadministered together with an immuno suppressive protein, antibody,nucleic acid, or chemotherapeutic agent.

Preferably, when used in combination with the endothelial growth factorinhibitor binding constructs of the present invention, the resultsobtained are synergistic. That is to say, the effectiveness of thecombination therapy of a binding construct and the immunosuppressiveagent is synergistic, i.e., the effectiveness is greater than theeffectiveness expected from the additive individual effects of each.Therefore, the dosage of the immunosuppressive compound can be reducedand thus, the risk of the toxicity problems and other side effects isconcomitantly reduced.

Any immunosuppressant therapy that has some efficacy at reducingtransplant rejection alone can be used in combination with theinhibitors of the invention, and such combinations are specificallycontemplated as combination therapies of the invention.

Corticosteroids are generally considered to be a first-line therapy foracute allograft rejection. Exemplary corticosteroids include prednisoneand prednisolone, which are commonly used for prophylaxis againstrejection, and methylprednisolone, which is often used for incidences ofacute rejection. Dosage of corticosteroids is well developed by those inthe field of transplant medicine, and varies depending on whetherprescribed as initiation or maintenance therapy, and depending onpatient tolerance. It is contemplated that reduced doses ofcorticosteroids may be used when combined with the inhibitors of theinvention. The lower dosing is expected to help reduce known adverseeffects of corticosteroids, which include hyperglycemia, diabetesmellitus, edema, hypertension, hyperlipidemia, hypokalemia, hirsutism,GI bleeding, arthralgia, osteoporosis, and psychosis.

A second class of immunosuppressants used with transplant patients, andcontemplated for combination of therapy of the invention, is calcineurininhibitors. Representative members of the class include cyclosporine andtacrolimus. These molecules act as immunosuppressive agents by bindingto immunophilin molecules to inhibit calcineurin, and thereby inhibit Tcell activation and proliferation. The patient's blood/serum and renaland liver functions are monitored to maintain an effective dose whileminimizing toxicity. Combination of cyclosporine with otherimmunosuppressants is known to cause side-effects and may require dosemodulation. The main side effects of CNIs are nephrotoxicity andneurotoxicity.

The mTOR inhibitors (mammalian target of rapamycin) represent a thirdclass of immunosuppressants. The mTOR inhibitors, which includesirolimus and everolimus, inhibit T cell proliferation. Sirolimus hasbeen used extensively in renal transplantation, and can be taken orally.Side effects associated with sirolimus therapy include dyslipidemia,hypertension, thrombocytopenia, anemia, peripheral edema, and tremor.

Antiproliferative agents represent a fourth class of immunosuppressants,and include azathioprine. A common starting dose for adults is 50 mg perday. After 4 to 8 weeks, the dose may be increased. Most adults require50-150 mg per day. A dose-dependent bone marrow suppression may occur inover half of patients treated with azathioprine, and hepatotoxicity hasbeen reported. Mycophenolic acid is another antiproliferative, whichtargets inosine monophosphate dehydrogenase (IMPDH), and causes arelatively selective suppression of lymphocyte proliferation. Comparedto many of the other immunosuppressants, MPA lacks significantnephrotoxicity. The capsules come in 250 mg and 500 mg and the usualdose for adults is 1-1.5 g (2-6 capsules) twice a day. The dose forchildren is 15 mg/kg/day.

A fifth class of immunosuppressants used in transplant patients isanti-lymphocyte-depleting antibodies: Induction therapy prior totransplantation and during the first weeks post-transplantation with anantilymphocyte-depleting antibody or an IL-2 receptor (IL-2R) antagonistcan provide effective protection against rejection. Polyclonalantilymphocyte-depleting antibodies, ATGAM and Thymoglobulin, sometimesare used after organ transplantation to reduce the risk of acuterejection. These antibodies are directed against T and B lymphocytes,and cause T cell lysis and blockade of B cell activation.

Muromonab C3 is a monoclonal antibody that is sometimes used in thetreatment of acute rejection in transplant recipients. It binds to CD3expressed on T cells and interferes with T cell antigen recognition.

IL-2R antagonists represent yet another class of immunosuppressants.Exemplary members of this class include basiliximab (Simulect) anddaclizumab (Zenopax). The act by binding to the CD25 subunit of theIL-2R, to block T cell activation. They are used for prophylaxis, ratherthan treatment, of acute rejection because during acute rejection, Tcells may become activated without the involvement of the CD25 subuniton the IL-2 receptor.

Mercaptopurine (6-MP) belongs to a group of drugs known asantimetabolites. It is used to treat many types of autoimmune diseases.It may interfere with the normal menstrual cycle in women and may stopsperm production in men. The usual adult dose is 2.5 mg/kg/day (100-200mg). The pediatric dose is 50 mg per day. A maintenance dosage afterremission is 1.5-2.5 mg/kg/day.

The foregoing list is not intended to be limiting. Inhibitors of theinvention can be co-administered with any immunosuppressive agent tobenefit from their complementary effects.

Other exemplary therapies to combine with therapies that targetendothelial growth factors and growth factor receptors include thosedescribed in International Application No. PCT/EP200 4/012406 (WO2005/049021), incorporated herein by reference in its entirety, relatingto a combination of an inhibitor of a mammalian Target of Rapamycin(mTOR), such as rapamycin; and an inhibitor of a Platelet-Derived GrowthFactor Receptor (PDGF-R), such as imatinib mesylate.

mTOR inhibitors include, but are not limited to the following drugs:

Brand Name Patent (U.S. Unless or Product # Generic Name Specified) orReference Rapamune ® rapamycin (sirolimus) 3,929,992; 5,288,711;5,516,781 RAD001 everolimus; 40-O-(2- EP663916; U.S. Pat. Appl.(Certican ®) hydroxyethyl)-rapamycin 20030170287 CCI-779 Rapamycin42-ester with 6,617,333; 5,362,718; 3-hydroxy-2- 6,277,983(hydroxymethyl)-2- methylpropionic acid Tumstatin and related U.S. Pat.Appl. 20030144481 polypeptides ABT578 U.S. Pat. Appl. 20030073737“rapalogs,” U.S. Pat. Appl. 20030073737; e.g., AP23573, WO01/02441;WO01/14387 AP22594 AP23841 ARIAD Pharmaceuticals TAFA93 Isotechnika

In addition to rapamycin and those derivatives of rapamycin listed inthe above table those discussed in U.S. Pat. Appl. No. 20030170287 mayalso be used. See also WO 94/09010, and WO 96/41807. Rapamycinderivatives may also include without limitation “rapalogs,” e.g., asdisclosed in WO 98/02441 and WO01/14387; deuterated rapamycin analogs,e.g., as disclosed in U.S. Pat. No. 6,503,921. Derivatives of other mTORinhibitors are also contemplated.

Exemplary Platelet-Derived Growth Factor Receptor (PDGF-R) inhibitorsinclude the following without limitation:

Brand Name Patent (U.S. Unless or Product # Generic Name Specified) orReference Gleevec imatinib mesylate 5,521,184; Druker, Nature(CGP57148B; STI-571) Medicine, 2: 561 (1996) AG1295 Kovalenko, CancerRes 54: 6106 (1994) AG1296 6,7-demethoxy-2- Kovalenko, Cancer Resphenylquinoxaline 54: 6106 (1994) AG1478 4(3-chlorophenyamino)- Lipson,K., et al., J. 6,7,-dimethoxyquina- Pharmacology and zoline ExperimentalTherapeutics, 285: 844-852 (1998) Trapidil triazolopyrimidine Lotinum etal., Endocrinol- ogy, 144: 2000-2007 (2003) 2-phenylaminopyrimi-Buchdunger et al., Cancer dine class compounds Res., 56: 100-1044 (1996)3744W Spacey et al., Bioch. Pharm. 55(3): 261-71, 1998 Feb. 1 TyrphostinKovalenko et al, Cancer Res. AG1296 1994 Dec 1; 54(23) CGP 79787DWO00/09098 CGP53′716 Buchdunger et al., PNAS 1995 92(7): 2558-62CGP57′148 Buchdunger et al., Cancer Res. 1996 56(1): 100-4 CT52923 U.S.Pat. Appl. No. 20030170287; Lokker et al., Cancer Res., 62: 3729-35(2002). RP-1776 U.S. Pat. Appl. No. 20030170287; Toki et al., J Antibiot(Tokyo). 54: 405- 14 (2001) GFB-111 U.S. Pat. Appl. No. 20030170287;Blaskovich et al., Nat Biotechnol. 18: 1065-70 (2000). a pyrrolo[3,4-c]-U.S. Pat. Appl. No. beta-carboline-dione 20030170287; Teller et al., EurJ Med Chem., 35: 413-27 (2000) SU11248 Sugen Pharmaceutical; (SU01248)Abrams et al., Mol Cancer Ther. 2: 1011-21 (2003); Mendel et al. ClinCancer Res. 9: 327-37 (2003). PKC787 Novartis PTK787 Novartis; U.S. Pat.Appl. No. 20030087934; Wood et al., Cancer Res. 60: 2178- 89 (2000).DMBI Organon; Zaman, Biochem. Pharmacol. 57: 57 1999 SU101 (LFM, Sugen;Shawner, Clin. HWA486) Cancer. Res., 3: 1167 1997 SU0020 Sugen; Zhang etal., J. (A771726) Pharm. Biomed. Anal. 28: 701-9 (2002). Comp. 54Parke-Davis; Boschelli, J. Med. Chem. 41: 4365 (1998)

The above list of PDGF-R inhibitors is not meant to be limiting. AnyPDGF-R inhibitor may be employed, including without limitation PDGF-Rinhibitors described in U.S. Pat. Nos. 5,932,580, 6,331,555, and6,358,954; WO 99/28304; WO 00/09098; WO 01/64200. Other inhibitors thatmay be used include 3-Substituted Indolin-2-ones (e.g., SU5416, SU6668),and derivatives thereof (Sun et al., J. Med. Chem., 41:2588-2603; Sun etal., J. Med. Chem. 43:2655-2663 (2000));2-Amino-8H-pyrido[2,3-d]pyrimidines (Boschelli et al., J. Med. Chem.41:4365-4377 (1998)).

In some embodiments, the PDGF-R inhibitor is a compound described inU.S. Pat. No. 5,521,184, incorporated herein by reference.

In addition to or in substitution for PDGF-R inhibitors, inhibitors ofother tyrosine kinases (receptors and other types as well) may also beused in accordance with this invention. Some of these inhibitors mayinhibit multiple kinases including, but not limited to, PDGF-Rs.Appropriate TK inhibitors are also taught in WO 99/03854; WO 01/64200;U.S. Pat. No. 5,521,184; WO 00/42042; WO 00/09098; EP 0 564 409 B1; U.S.Pat. No. 5,521,184; WO 97/32604; U.S. Pat. No. 6,610,688; US PatentAppl. Pub. No. 20030194749; Livitzki, A., et al., “Protein TyrosineKinase Inhibitors as Novel Therapeutic Agents, “Pharmacol. Ther.82:231-29 (1999). Other classes of compounds may also be employed. Forexample and without limitation, Leflunomide (U.S. Pat. No. 4,284,786)and/or derivative FK778 may be used. (See, e.g., Savikko Transplantation2003:76:455 and editorial Williams ibid p 471.)

E. Suitable Transplant Recipients

The present invention is applicable to all cell, tissue, organ fragment,organ, and multi-organ transplant procedures, to inhibit or delayrejection reactions or other undesired side-effects, includingarteriosclerosis, that are associated with transplants. Thus, theinvention is applicable to any mammal that receives any cell, tissue,organ fragment, organ, or multi-organ transplant.

Exemplary transplants include autografts (transplant or transfer oftissue from an organism to itself, e.g., where donor and recipient arethe same organism); isografts (transplants from a donor organism to agenetically identical recipient (e.g., an identical twin or a clone);allografts (transplants from a donor organism to a geneticallynon-identical organism of the same species); and xenografts (transplantsof organs or tissue from one species to another). All of these types oftransplants are theoretically possible in humans, although xenograftshave thus far been fairly limited (e.g., heart valves), and isograftdonors are quite rare. Thus, in the context of transplantation of vitalorgans or tissues to replace diseased ones, allograft transplantsrepresent the most common class for human therapy. The likelihood ofrejection or graft arteriosclerosis increases as the differences betweendonor and recipient increase.

Exemplary organs that have been transplanted in humans, and for whichthe methods of the invention are especially applicable, include thoracicorgans (e.g., heart, lung); abdominal organs (e.g., liver, kidney,pancreas, small intestine). The invention also is applicable for varioustissue and cell transplants, including but not limited to pancreaticislet cells, bone marrow cells, cardiac myocytes, blood vessels orvessel fragments, heart valves, bones, and skin. The invention also isapplicable to the emergent fields of transplantation of embryonic stemcells and various pluripotent or multipotent progenitor or precursorcells that have the potential to differentiate into one or more celltypes (e.g., hematopoietic progenitor/stem cells, neural progenitor/stemcells, endothelial progenitor cells, and muscle progenitor cells).

F. Transplant Rejection and Monitoring of Transplanted Tissue

After surgery, the transplanted cell, tissue or organ is carefullymonitored for any signs that the recipient will reject the new cell,tissue or organ. There are three main types of transplant rejections:(1) hyperacute, (2) acute, and (3) chronic transplant rejections.

Hyperacute rejection is a complement-mediated response in recipientswith pre-existing antibodies to the donor (for example, ABO blood typeantibodies). Hyperacute rejection occurs within minutes and thetransplant must be immediately removed to prevent a severe systemicinflammatory response. Rapid coagulation of the blood occurs. This is aparticular risk in kidney transplants, and so a prospective cytotoxiccrossmatch is performed prior to kidney transplantation to ensure thatantibodies to the donor are not present. For other organs, hyperacuterejection is prevented by transplanting only ABO-compatible grafts.Hyperacute rejection is the likely outcome of xenotransplanted organs.

Acute rejection is generally acknowledged to be mediated by T cellresponses to proteins from the donor organ, which differ from thosefound in the recipient. Unlike antibody-mediated hyperacute rejection,development of T cell responses first occurs several days after atransplant if the patient is not taking immunosuppressant drugs. Sincethe development of powerful immunosuppressive drugs, such as thosediscussed above, the incidence of acute rejection has been greatlydecreased. However, organ transplant recipients can develop acuterejection episodes months to years after transplantation. Acuterejection episodes can destroy the transplant if it is not recognizedand treated appropriately. A single episode is not a cause for graveconcern if recognized and treated promptly, and rarely leads to organfailure, but recurrent episodes are associated with chronic rejection ofgrafts.

The bulk of the immune system response is to the MajorHistocompatibility Complex (MHC) proteins. MHC proteins are involved inthe presentation of foreign antigens to T cells, and receptors on thesurface of the T cell (TCR) are uniquely suited to recognition ofproteins of this type. MHC are highly variable between individuals, andtherefore the T cells from the donor recognize the foreign MHC with avery high frequency, leading to powerful immune responses that causerejection of transplanted tissue. Identical twins and cloned tissue areMHC matched, and are therefore not subject to T cell mediated rejection.

The term “chronic rejection” is used when the process is shown to be dueto a chronic alloreactive immune response. It can be caused by a memberof the Minor Histocompatibility Complex such as the H—Y gene of the maleY chromosome, and usually leads to the need for a new organ after adecade or so.

Cardiac Allograft vasculopathy (CAV), also known as chronic cardiacrejection or transplant coronary artery disease, is the main factorlimiting the long term success of heart transplants, and most likelyinvolves both immunological and non-immunological factors (Al-Khaldi etal., Annu. Rev. Med., 57:455-471, 2006, the disclosure of which isincorporated herein by reference). Identified risk factors include olderdonor and recipient age, ischemia-reperfusion injury, human leukocyteantigen (HLA) mismatch, hypertension, hyperlipidemia, insulinresistance, cytomegalovirus infection, and recurrent rejection (Tayloret al., J. Heart Lung Transplant., 23:796-803, 2004; Valantine et al.,Circ., 103:2144-2152, 2001; Costanzo-Nordin et al., J. Heart. LungTransplant., 11:S90-103, 1992; Grattan et al., JAMA, 261:3561-3566,1989; Kobashigawa et al., J. Heart Lung. Transplant., 14:S221-S226,1995; and Valantine, H., J. Heart Lung. Transplant., 23:S187-S193,2004). These risk factors may lead directly or indirectly to endothelialinjury with subsequent intimal hyperplasia and vascular smooth muscleproliferation.

Monitoring of heart transplant recipients for the development ofallograft rejection includes non-invasive methods such asintramyocardial electrocardiogram (Hetzer et al., Ann Thorac Surg66:1343, 1998) and echocardiography, radioisotope techniques, magneticresonance imaging and immunological methods (Kemkes et al., J Heart LungTransplant 11: S221-31, 1992). The immunological methods include themeasurement of serum cytokine levels, particularly IL6 and IL8 (Kimballet al., Transplantation 61: 909-15, 1996), monitoring recipient serumfor donor HLA antigens and anti-HLA antibodies (Reed et al.,Transplantation 61: 566-72, 1996), and measuring reactivity ofallo-reactive helper T cells (DeBruyne, Transplantation 56: 722-7,1993), or cytotoxic T cells in the blood of the recipient (Reader et al.Transplantation 50: 29-33, 1990; Loonen et al., Transplant Int7:596-598, 1994). Invasive methods include an endomyocardial biopsy,which detects an ongoing immune rejection process that may have alreadydamaged the heart before immunosuppressive intervention has beeninitiated. Non-invasive methods are associated with the detection ofongoing damage in the heart muscle and thus, may come too late for thepreferred goal in improving the care of the transplant recipient: earlyprevention of the developing rejection episode.

The monitoring methods used in other organ allograft recipients are alsodirected to detection of damage to the transplanted organ. With respectto kidney allograft recipients, noninvasive methods include thefunctional indicators of impaired renal activity, such as (a) decreasedurine volume, (b) decreased clearance of creatinine and (c) elevatedblood urea nitrogen. Monitoring includes detection of lymphocytes in theurine (Salaman, Immunol Lett 29: 139-12, 1991), secretion of neopterin(a pteridine from stimulated macrophages) and interferon-gamma (acytokine released by activated T cells) (Khoss et al. Child Nephrol Urol9:46-49, 1988; Grebe et al., Curr Drug Metabol 3:189-202, 2002). Thesensitivity of detection of inflammatory products in the urine wasfurther improved by measuring the presence of mRNA for perforin andgranzyme B (proteins released from T cells that damage target cells),using the reverse transcriptase-polymerase chain reaction (RT-PCR) foramplification and detection of these molecules (Li et al., N Engl J Med344: 947-954, 2001).

V. NON-EXCLUSIVE EXAMPLES OF THE INVENTION

The invention may be more readily understood by reference to thefollowing examples, which are given to illustrate the invention and notin any way to limit its scope. The first several examples describemaking and testing inhibitor compounds useful to practicing methods ofthe invention. The second group of examples describe evidence that theantigens are suitable targets for therapy and/or evidence that suchtherapy is efficacious. These examples primarily make reference tobinding constructs that bind particular growth factors of the VEGFsubfamily, but they may also be adapted for use of binding constructsthat bind other VEGF subfamily members, as well as for bindingconstructs that bind PDGF subfamily members. Similarly, bindingconstructs comprising other VEFGR receptor fragments, PDGFR receptorfragments, and neuropilin receptor fragments may also be employed invariations of these examples.

Example 1 VEGFR-2 and VEGFR-3 Fragments that Bind VEGF-A or VEGF-C

To determine the portion of a receptor's extracellular domain (ECD) thatwas sufficient for ligand binding, fragments of the ECDs of VEGFR-2(R-2) and VEGFR-3 (R-3) were used to make various soluble constructs.The constructs included Fc domain human IgG fragments fused to theC-terminus of the receptor fragments. As indicated in Tables 3 and 4,some constructs were made using a heterologous (N-terminal) signalpeptide derived from CD33.

Construction of Fragments and Plasmids

R-2 Constructs

To construct the VEGFR-2/IgG expression plasmid, the construct, R-2 A,comprising the first three Ig-domains (D1-3) of VEGFR-2 was amplified byPCR using primers 5′-GCGGATCCTTGCCTAGTGTTTCTCTTGATC-3′ (SEQ ID NO: 72),and 5′-CCAGTCACCTGCTCCGGATCTTCATGGACCCTGACAAATG-3′ (SEQ ID NO: 73), andcloned into the Signal plgplus vector (Novagen, Madison, Wis.). Theresulting plasmid was digested with BamHI and KpnI, treated with T4polymerase and back-ligated. To assemble other VEGFR-2/IgG constructs,PCRs were performed using the D1-3 construct as the template, T7 forwardprimer and the following reverse primers:

(R-2 F), (SEQ ID NO: 59) 5′-GCTGGATCTTGAACATAGACATAAATG-3′, (R-2 B),(SEQ ID NO: 60) 5′-CTAGGATCCCCTACAACGACAACTATG-3′, (R-2 C), (SEQ ID NO:61) 5′-CTAGGATCCACATCATAAATCCTATAC-3′, (R-2 D), (SEQ ID NO: 62)5′-GCATGGTCTCGGATCATGAGAAGACGGACTCAGAAC-3′, (R-2 E) (SEQ ID NO: 63)5′-CTAGGATCCTTTTCTCCAACAGATAG-3′; forward primer (SEQ ID NO: 64)5′-AGCGCTAGCGTTCAAGATTACAGATCTCC-3′, and the following reverse primers:(R-2 G), (SEQ ID NO:65) 5′-ATGTGTGAGGTTTTGCACAAG-3′, (R-2 H), (SEQ IDNO: 66) 5′-CTAGGATCCCCTACAACGACAACTATG-3′, (R-2 I), (SEQ ID NO: 67)5′-CTAGGATCCACATCATAAATCCTATAC-3′, (R-2 J), (SEQ ID NO: 68)5′-GCATGGTCTCGGATCATGAGAAGACGGACTCAGAAC-3′, (R-2 K), (SEQ ID NO: 69)5′-CTAGGATCCTTTTCTCCAACAGATAG-3′, forward primer (SEQ ID NO: 70)5′-AGCGCTAGCTATAGGATTTATGATGTG-3′, and reverse primer (R-2 L), (SEQ IDNO: 71) 5′-ATGTGTGAGGTTTTGCACAAG-3′.

The PCR products were digested with NheI and BstYI (R-2 F and Lconstructs), NheI and BamHI (R-2 E, and H-K constructs), BamHI (R-2linker B and C constructs), BamHI and BsaI (R-2 D construct), or NheIand BsmBI (R-2 G construct), and cloned into the Signal plgplus vector.In order to repair frame-shifts in constructs containing nucleotidesequence coding for domain 1 of VEGFR-2, the vectors were cut withrestriction enzyme NotI, blunted with Klenow enzyme, cut with EcoRV andback-ligated.

R-3 Constructs

A series of R-3 constructs with C-termini between Ig domains 2 and 3 ofVEGFR-3 (R-3 C through F constructs) was created by PCR using theexpression plasmid comprising the R-3 D1-3 transcript (e.g., the R-3 Gconstruct, SEQ ID NO: 43) as template, T7 as forward primer and thefollowing reverse primers:

5′-TCAGGATCCGCGAGCTCGTTGCCTG-3′, (SEQ ID NO: 74)5′-TACAGGATCCCCTGTGATGTGCACCAG-3′, (SEQ ID NO: 75)5′-TCAGGATCCGCGTGCACCAGGAAGG-3′, (SEQ ID NO: 76) and5′-TCAGGATCCGCGAAGGGGTTGGAAAG-3′. (SEQ ID NO: 77)

The Ig homology domain 1 was deleted from the D1-3 expression plasmid(R-3 G construct) by site-directed mutagenesis using primers

5′CCTTGAACATCACGGAGGAGTCACACGTCAGAGACTTTGAGCA GCCATTCATCAACAAGC-3′ (SEQID NO: 78) and

5′ AGCTGCTGGTAGGGGAGAAGGATCCTGAACTGCACCGTGTGG-3′ (SEQ ID NO: 79), andexcision of the BamH I fragment from the resulting plasmid. Thatprocedure combined with the described truncation primers, for R-3 Cthrough F constructs, allows for the production of the R-3 constructs(e.g., C, D, E, F, J, K, L, and M). The plasmid coding for domains 2 and3 of VEGFR-3 (R-3 I) was made by transfer of the Sph I fragment from theoriginal expression R-3 D1-3 plasmid into the plasmid encoding onlydomain 2 of VEGFR-3 (R-3 J). The sequence derived from a particularreceptor is listed in Table 2. Expression was performed using standardcalcium phosphate-mediated transfection into 293T cells.

The binding assays utilized minimal VEGF-A (SEQ ID NOS: 106 and 107) andVEGF-C (SEQ ID NOS: 108 and 109) fragments with 109 residues each(called VEGF-A 109 and VEGF-C 109). These constructs are not naturallyoccurring, but are effective for binding assays. Other growth factorconstructs, either natural or artificial, may also be used forperforming these assays.

Either Tritiated VEGF-A 109 or VEGF-C 109 was used in a given bindingexperiment. Ligand in solution was precipitated by mixing 175 μl ofligand solution with 100 μl binding mix at 4° C. overnight, withagitation. The ligand solution may be the supernatant of metabolicallylabeled 293T cells. The binding mixes used for the receptor bindinganalysis were as follows: for VEGFR-1 binding assays, the binding mixwas phosphate buffered saline (PBS) containing 1.5% BSA, 0.06% Tween 20,3 μg/ml heparin and 400 ng/ml VEGFR-1-Fc fusion protein (100 μl of thisbinding mix was added to 200 μl of ligand solution). For VEGFR-2 bindingassays, the binding mix was 82% conditioned cell supernatant from 293Tcells transiently expressing VEGFR-2-Fc fusion protein in mixture with18% of a PBS solution that contained 5% BSA, 0.2% Tween 20, and 10 μg/mlheparin (250 μl of binding mix was added to 200 μl of ligand solution).For VEGFR-3 binding assays, the binding mix was 82% conditioned cellsupernatant from 293T cells transiently expressing VEGFR-3-Fc fusionprotein, 18% of PBS containing 5% BSA, 0.2% Tween 20, and 10 μg/mlheparin (250 μl of binding mix was added to 200 μl of ligand solution).To collect precipitated ligand, 50 μl of a 30% protein A sepharose (PAS,Pharmacia) slurry in PBS was added and incubated under agitation for atleast 1.5 hr at 4° C. Standard buffer was added to eachimmunoprecipitation sample and boiled for 5 minutes at 95° C. duringwhich the immunopreciptated proteins become dissociated from the proteinA sepharose. After centrifugation, 10 μl of each sample was analyzed on15% SDS-PAGE under reducing conditions. The gels were dried and exposedfor either 12 hours on phosphorimager plates or 4 weeks on X-ray film.

Tables 3 and 4 identify constructs by name, a DNA and deduced amino acidsequence from the sequence listing, the portion of VEGFR-2 (SEQ ID NO:4) or VEGFR-3 (SEQ ID NO: 6) amino acid sequence that was included inthe constructs, whether the constructs expressed, and, if tested,whether constructs bound ligand. The table data is compiled fromanalysis of PAGE gels. The asterisk adjacent to the “B*” indicates a“spill-over” from the adjacent lane, as the origin of the bands seen inthe “B” lane. A failure to express under the particular experimentalconditions used in this instance should not be interpreted as a failureto bind. The experiments can be repeated using different receptorfragments, binding constructs, ligands, or combinations thereof.

TABLE 3 VEGFR-2 CONSTRUCTS Fc Fusion SEQ ID Binds Binds Constructs SEQID NOS: NO: 4 Expression VEGF-A VEGF-C R-2 A SEQ ID NOS:  24-326 Yes YesYes with CD33 Signal 7 and 8 Peptide R-2 B SEQ ID NOS:  24-220 Yes No Nowith CD33 Signal 9 and 10 Peptide R-2 C SEQ ID NOS:  24-226 Yes No Nowith CD33 Signal 11 and 12 Peptide R-2 D SEQ ID NOS:  24-232 Yes No Nowith CD33 Signal 13 and 14 Peptide R-2 E SEQ ID NOS:  24-241 Yes No Nowith CD33 Signal 15 and 16 Peptide R-2 F SEQ ID NOS:  24-122 Yes No Nowith CD33 Signal 17 and 18 Peptide R-2 G SEQ ID NOS: 118-326 Yes Yes Yeswith CD33 Signal 19 and 20 Peptide R-2 H SEQ ID NOS: 118-220 Yes No Yeswith CD33 Signal 21 and 22 Peptide R-2 I SEQ ID NOS: 118-226 Yes No Weakwith CD33 Signal 23 and 24 Peptide R-2 J SEQ ID NOS: 118-232 Yes No Verywith CD33 Signal 25 and 26 Weak Peptide R-2 K SEQ ID NOS: 118-241 Yes NoNo with CD33 Signal 27 and 28 Peptide R-2 L SEQ ID NOS: 220-326 Yes NoNo with CD33 Signal 29 and 30 Peptide

TABLE 4 VEGFR-3 CONSTRUCTS Fc Fusion Sequence ID SEQ ID Binds ConstructsNos. NO: 6 Expression VEGF-C R-3 A with CD33 SEQ ID NOS: 138-329 Yes —Signal Peptide 31 and 32 R-3 B with CD33 SEQ ID NOS: 138-226 Yes YesSignal Peptide 33 and 34 R-3 C SEQ ID NOS: 1-229 Yes Yes 35 and 36 R-3 DSEQ ID NOS: 1-226 Yes Yes 37 and 38 R-3 E SEQ ID NOS: 1-223 No — 39 and40 R-3 F SEQ ID NOS: 1-220 No — 41 and 42 R-3 G SEQ ID NOS: 1-329 YesYes 43 and 44 R-3 H SEQ ID NOS: 1-134 Yes No 45 and 46 R-3 I SEQ ID NOS:1-39, Yes No 47 and 48 132-329 R-3 J SEQ ID NOS: 1-39, Yes No 49 and 50132-247 R-3 K SEQ ID NOS: 1-39, Yes No 51 and 52 132-229 R-3 L SEQ IDNOS: 1-39, Yes — 53 and 54 132-226 R-3 M SEQ ID NOS: 1-39, No — 55 and56 132-223 R-3 N SEQ ID NOS: 1-40, — — 57 and 58 226-329

The results of these assays demonstrate that novel receptor fragmentsare capable of binding ligands that the receptor as a whole may bind. Inaddition to providing a clearer picture as to what regions of the ECDare necessary for ligand binding, the binding data identifies receptorfragments useful as therapeutics.

The present data show that the R-2H fragment of R-2 of approximately 100residues and spanning D2 of R-2 is sufficient for VEGF-C binding. ForR-3, a larger fragment is required for VEGF-C binding, e.g., the R-3 Dconstruct in table 4, which spans D1-2 of R-3.

Three-dimensional modeling based on the structure of VEGFR-1 complexedwith VEGF-A was used to predict that a groove in VEGF-C mightaccommodate the region between Ig-like domains 2 and 3 of VEGFR-3(Flt4). WO 01/62942. The present data shows for the first time thatsequence intermediate between the second and third Ig domains of R-3 isimportant for ligand binding.

For R-1 and R-2, the first Ig-domain has been described as inhibitoryfor VEGF-A binding. Lu, et al., J. Biol. Chem., 275(19): 14321-14330(2000); Shinkai, A. et al., J. Biol. Chem., 273(47):31283-88 (1998). ForVEGF-C binding, the present data show that the inhibitory role of thefirst Ig-domain appears to apply to R-2 fragments, but not R-3fragments.

The data also provides novel information regarding R-2 fragments andVEGF-A binding. Conflicting reports exist for constructs comprising thesecond and third Ig-domains of R-2 and VEGF-A binding. Fuh, et al., J.Biol. Chem., 273(18): 11197-11204 (1998); Niwa, et al., U.S. Pat. No.6,348,333; Shinkai, A. et al., J. Biol. Chem., 273(47):31283-88 (1998).Fuh reported that only domains 2 and 3 were needed. Niwa taught thatonly 1 and 2 were needed. Shinkai stressed the importance of domain 4 ofR-2. The issue is further confused because different reports havedefined the boundaries of the Ig-domains in different ways, i.e.,different start and stop points, a practice that has been recognized aspotentially affecting whether fragments bind ligands, and with whatdegree of affinity. Shinkai, A. et al., J. Biol. Chem., 273(47):31283-88(1998).

Example 2 Ligand Binding Assays Involving Binding Constructs with Morethan One Binding Element

The assays as performed in Example 1 are repeated, substituting abinding construct with multiple binding units. For example, one employsa binding construct comprising a binding unit that binds VEGF-A and abinding unit that binds VEGF-C. One looks for the ability of such abinding construct to bind both VEGF-A and VEGF-C. This information maybe obtained by using different radio- or other labels, e.g., fluorescentlabels for fluorescence resonance energy transfer (FRET), on each typeof ligand or use of labels on the binding construct and or ligands, todetermine whether a given binding construct molecules are binding amolecule of VEGF-A and VEGF-C. Constructs that are shown to bind morethan one growth factor ligand, as well as those described in Example 1and elsewhere herein, have an indication for anti-neoplastic therapieswhere multiple growth factors contribute to neoplastic cell growth.

Example 3 Chimeric VEGFR Binding Constructs which Bind Multiple Ligands

As stated above, constructs that bind more than one growth factor ligandhave an indication as anti-neoplastic therapies where multiple growthfactors contribute to neoplastic cell growth. In order to determine theefficacy of a binding construct designed to bind more than one growthfactor, two chimeric binding constructs were generated and their abilityof each to bind to two growth factors was measured.

The binding constructs were designed as immunoblobulin fusion proteinsas described above. To construct chimeric VEGF receptor/hIgG1Fc fusionproteins, the pIgPlus vector was used to build a construct comprisingthe first immunoglobulin-like domain of VEGFR-3 and the second and thirdIg-like domains of VEGFR-2. The construct is designatedR-3D1-R2D2+3/hIgG1Fc. To clone the R-3D1-R2D2+3/hIgG1Fc construct, PCRwas performed with CMV forward primer (18782, 5′TACTTGGCAGTACATCTACGTATTAGTCATCGC-3′) (SEQ ID NO: 122) and reverseprimer v360 (5′-CGGAGATCTGTAGTCTTGCACGTACACGTAGGAGCTGGC-3′) (SEQ ID NO:123) using plgPlus-hVEGFR-3D1-3-IgG1Fc as a template. The PCR-productwas cut with SnaBI and BglII. The 718 bp D1-R2D2+3/hIgG1Fc insert wasligated into the SnaBI- and partially BglII-cut vectorplgPlus-hVEGFR-2D1-3-IgG1Fc described above. The presence and sequenceof the correct insert was confirmed by sequencing a representativeisolated hVEGFR-3D1-R2D2+3/hIgG1Fc clone (clone #2). (SEQ ID NO: 124 andSEQ ID NO: 125).

In addition to the above chimeric construct, a chimeric VEGFreceptor/hIgG1Fc fusion protein was constructed having the first Ig-likedomain of VEGFR-3, the second Ig-like domain of VEGFR-2 and the thirdIg-like domain of VEGFR-1. The construct is designatedR-3D1-R2D2-R1D3/hIgG1Fc.

To clone the pIgPlus-hVEGFR-3D1-R2D2-R1D3/hIgG1Fc construct, PCR wasperformed using pIgPlus-hVEGFR-3D1-R2D2+3/hIgG1Fc as a template and theT7 forward and reverse primer v362(5′-TACAATTGAGGACAAGCGTATGTCCACGAAGTAGTTTAACTGGACGAGGCGTGCTTATTTGCACATCATAAATCCTATACC-3′) (SEQ ID NO: 126). The PCR-productwas cut with HindIII and MfeI/MunI. The 787 bp VEGFR-3D1-R2D2+3/hIgG1Fcinsert was ligated into the HindIII- and partially MfeI-cut vectorplgPlus-hVEGFR-1D1-3-IgG1Fc. The presence and sequence of the correctchimeric insert was confirmed by sequencing the a representativehVEGFR-3D1-R2D2-R1D3/hIgG1Fc clone (clone #6) (SEQ ID NO: 127 and SEQ IDNO: 128).

Expression of Chimeric VEGFR/hIgG1Fc Fusions:

For expression analysis, the two new chimeric VEGF receptors and controlconstructs expressing R-1D1-3/hIgG1Fc, R-2D1-3/hIgG1Fc, R-3D1-3/hIgG1Fc,mature VEGF-C and VEGF-A₁₆₅ were transiently transfected into 293T cellsusing JetPEI (QBioGene/MP Biomedicals, Irvine, Calif.). Metaboliclabeling with ³⁵S-methionine and ³⁵S-cysteine was carried out at 48hours post-transfection and labeling maintained for 24 hours. Theserum-free conditioned medium was then immunoprecipitated using ProteinA sepharose and either: a) specific antiserum against human matureVEGF-C; b) goat polyclonal antibody against human VEGF-A (R&D systems,Minneapolis, Minn.); or, c) serum-free medium of 293T cells taken 48 to72 hours post-transient transfection with VEGF receptor/hIgG1Fc proteins(control proteins, R-1D1-3, R-2D1-3, R-3D1-3; chimeric proteins,R-3D1-R2D2+3 and R-3D1-R2D2-R1D3).

The immunoprecipitated fractions were analyzed on 17% SDS-PAGE and thedried gels were exposed for 12 hours on phosphoimager plates or 36 hourson X-ray films. Expression analysis demonstrated that the chimericreceptor fusion proteins exhibited high expression levels in transfected293 T cells.

Analysis of Binding Properties of Chimeric VEGF Receptor/hIgG1FcFusions:

Ligand binding analysis was performed as described for the VEGF-C/VEGF-Ahybrid growth factors in Example 1. Briefly, the unlabeled conditionedmedium of transiently transfected 293T cells expressing the chimericVEGFR/IgG1Fc fusion proteins was used to precipitate the ³⁵Smetabolically labeled mature VEGF-C, full-length VEGF-C, and VEGF-A₁₆₅.SDS-PAGE of ligands immunoprecipitated with chimeric and controlVEGFR/IgFc showed that the R-3D1-R2D2-R1D3/Ig chimeric protein stronglybound both VEGF-A and VEGF-C, as predicted based on the VEGFR2 and R1immunoglobulin domains. In one experiment, the chimeric constructR-3D1-R2D2+3/Ig exhibited binding to VEGF-C and not VEGF-A. A secondexperiment with the R-3D1-R2D2+3/Ig construct showed only weak bindingto VEGF-A.

These results demonstrate that the ligand binding constructs generatedherein are useful in developing compositions that bind multiple growthfactors involved in numerous cell activities. These constructs providepromising therapy for diseases such as cancer and other proliferativediseases wherein multiple growth factors mediate the condition ordisease state.

Example 4 Assay for Neutralization of Growth Factor Activity

The following protocol provides an assay to determine whether a bindingconstruct neutralizes one or more PDGF/VEGF growth factors by preventingthe growth factor(s) from stimulating phosphorylation of its receptor.

Cells such as NIH 3T3 cells are transformed or transfected with a cDNAencoding a PDGFR/VEGFR receptor, such as VEGFR-3, and cultured underconditions where the encoded receptor is expressed on the surface of thecells. Transfected cells are cultured with either 1) plain growthmedium; 2) growth medium supplemented with 50 ng/ml of one or moreligands for the recombinant receptor, such as fully processed VEGF-Cand/or VEGF-D, which are ligands for VEGFR-3; 3) growth mediumsupplemented with 50 ng/ml of growth factor that does not bind therecombinant receptor (e.g., VEGF-A in the case of VEGFR-3), to serve asa control; or any of (1), (2), or (3) that is first pre-incubated withvarying concentrations of a binding construct to be tested.

After culturing with the culture mediums described above in the presenceor absence of the binding construct, the cells are lysed,immunoprecipitated using anti-receptor (e.g., anti-VEGFR-3) antiserum,and analyzed by Western blotting using anti-phosphotyrosine antibodies.Cells stimulated with the appropriate growth factor ligand (VEGF-C/D)stimulate VEGFR-3 autophosphorylation, which is detected with theanti-phosphotyrosine antibodies. Binding constructs that reduce oreliminate the ligand-mediated stimulation of receptor phosphorylation(e.g., in a dose-dependent manner) are considered neutralizing bindingconstructs.

Example 5 EPO Chimera Survival/Proliferation Blocking Assay

A binding construct is tested for the ability to block the binding ofthe growth factor(s) to their receptors, using bioassays of receptorbinding and cross-linking. These assays involve the use of Ba/F3 pre-Bcells which have been transfected with plasmid constructs encodingchimeric receptors consisting of the extracellular domain of growthfactor receptors and the cytoplasmic domain of the erythropoietinreceptor (Stacker, S A. et al., J. Biol. Chem. 274:34884-34892, 1999;Achen, M G. et al., Eur. J. Biochem. 267:2505-2515, 2000). These cellsare routinely passaged in interleukin-3 (IL-3) and will die in theabsence of IL-3. However, if signaling is induced from the cytoplasmicdomain of the chimeric receptors, these cells survive and proliferate inthe absence of IL-3. Such signaling is induced by ligands which bind andcross-link the extracellular domains of the chimeric receptors.Therefore binding of a growth factor ligand to the extracellular domainsof the chimeric receptors causes the cells to survive and proliferate inthe absence of IL-3. Addition of binding constructs that block thebinding of growth factor to the extracellular domains will cause celldeath in the absence of IL-3. An alternative Ba/F3 cell line whichexpresses a chimeric receptor containing the extracellular domain of theTie2 receptor (that does not bind VEGF family members) is not induced bythe relevant growth factors to proliferate and is used, in the presenceof IL-3, as a control to test for non-specific effects of potentialinhibitors.

In an exemplary assay, a binding construct that can bind VEGF-A andVEGF-C is tested. Samples of purified VEGF-A and VEGF-C are incubatedwith varying amounts of the binding construct for one hour at 4° C. inPBS before dilution of the mixtures 1:10 with IL-3-deficient cellculture medium. Ba/F3 cell lines expressing receptor(s) capable ofbinding the growth factors are then incubated in the media for 48 hoursat 37° C. To measure DNA synthesis in the cells, 1 μCi of 3H-thymidineis added and the cells are incubated for 4 hours prior to harvesting.Incorporated 3H-thymidine is measured using a cell harvester (Tomtec®)and beta counting. The ability of the binding construct to block growthfactor-mediated cell growth and survival (as measured by DNA synthesis)is analyzed relative to the control Tie2 cell line in the presence ofIL-3. Growth inhibition in the experimental group relative to thecontrol group demonstrates that the binding construct blocks cellgrowth, presumably by blocking the binding and cross-linking ofreceptors by growth factor ligands at the cell surface.

Example 6 Effect of Binding Constructs on BCE Migration

Solutions containing growth factors pre-incubated alone or with varyingconcentrations of a binding construct are placed in wells made incollagen gel and used to stimulate the migration of bovine capillaryendothelial (BCE) cells in the gel as follows. A further controlcomprising neither growth factor ligand nor binding construct may alsobe employed, as may a control with just binding construct. Bindingconstructs that cause a decrease in migration (relative to when growthfactor alone is employed) have an indication as therapeutics to preventor retard angiogenesis.

BCE cells (Folkman et al., Proc. Natl. Acad. Sci. (USA), 76:5217-5221(1979)) are cultured as described in Pertovaara et al., J. Biol. Chem.,269:6271-74 (1994). These or other cells employed may be transformedwith growth factor receptor if not already expressed. For testing ofVEGF-A/VEGF-C binding constructs, cells would be transformed with bothVEGFR-2 and/or VEGFR-3. The collagen gels are prepared by mixing type Icollagen stock solution (5 mg/ml in 1 mM HCl) with an equal volume of2×MEM and 2 volumes of MEM containing 10% newborn calf serum to give afinal collagen concentration of 1.25 mg/ml. The tissue culture plates (5cm diameter) are coated with about 1 mm thick layer of the solution,which is allowed to polymerize at 37° C. BCE cells were seeded on top ofthis layer. For the migration assays, the cells are allowed to attachinside a plastic ring (1 cm diameter) placed on top of the firstcollagen layer. After 30 minutes, the ring is removed and unattachedcells are rinsed away. A second layer of collagen and a layer of growthmedium (5% newborn calf serum (NCS)), solidified by 0.75% low meltingpoint agar (FMC BioProducts, Rockland, Me.), are added. A well (3 mmdiameter) is punched through all the layers on both sides of the cellspot at a distance of 4 mm, and the sample or control solutions arepipetted daily into the wells. Photomicrographs of the cells migratingout from the spot edge are taken after six days through an Olympus CK 2inverted microscope equipped with phase-contrast optics. The migratingcells are counted after nuclear staining with the fluorescent dyebisbenzimide (1 mg/ml, Hoechst 33258, Sigma).

The number of cells migrating at different distances from the originalarea of attachment towards wells containing sample solutions aredetermined 6 days after addition of the media. The number of cellsmigrating out from the original ring of attachment is counted in fiveadjacent 0.5 mm×0.5 mm squares using a microscope ocular lens grid and10× magnification with a fluorescence microscope. Cells migratingfurther than 0.5 mm are counted in a similar way by moving the grid in0.5 mm steps. The experiments are carried out twice with similarresults. Daily addition of 1 ng of FGF2 into the wells may be employedas a positive control for cell migration.

Example 7 Suppression of VEGFR-3 Signalling Pathway Improves AllograftSurvival

Experiments described herein elucidate the role of lymphatic vessels andtheir principal growth signalling pathway, VEGF-C/VEGFR-3, inexperimental cardiac allograft alloimmunity and arteriosclerosis. Wefound functional lymphatic vessels in rat cardiac allografts thatco-expressed LYVE-1, Prox-1, VEGFR-3 and chemokine CCL21, and wereactive in transferring antigen presenting cells (APC). Chronic rejectionenhanced the number of graft-infiltrating VEGF-C⁺ inflammatory cells,and induced myocardial lymphangiogenesis. Lymphatic EC almostexclusively originated from donor-derived cells. Systemic VEGFR-3inhibition with VEGFR-3-Ig gene delivery reduced allograft CCL21production, alloimmune activation, and improved cardiac allograftsurvival of recipients receiving suboptimal cyclosporine Aimmunosuppression. In a mouse chronic rejection model, treatment withneutralizing VEGFR-3 antibodies reduced allograft CCL21 production,inflammation and arteriosclerosis. Collectively, our results indicateinterplay of inflammation and lymphangiogenesis in cardiac allografts.Moreover, VEGFR-3 inhibition reduced APC trafficking possibly throughdirect DC-mediated and indirect CCL21 mediated effects. VEGFR-3inhibition may thus be used as a novel non-T cell-targeted inductiontherapy to regulate alloimmune activation after solid organtransplantation.

Methods Experimental Design

The effect of heart transplantation on the expression of lymphaticendothelial markers and lymphatic growth factors was investigated usinga rat heterotopic heart transplantation model comparing non-transplantedhearts, acutely and chronically-rejecting cardiac allografts, andsyngenic controls. Marker gene transgenic mice were used to determinethe origin of VEGFR-3⁺ lymphatic EC in chronically-rejecting mousecardiac allografts. The functional role of VEGFR-3 singalling inalloimmune responses was investigated by perfusing rat cardiac allograftrecipients intrapotally with an adenovirally expressed soluble VEGFR-3receptor extracellular domain (Ad.VEGFR-3-Ig) that traps VEGFR-3ligands. Neutralizing monoclonal VEGFR-3 antibodies (VEGFR-3 mAb) wereused to confirm the effect of VEGFR-3 inhibition on lymphangiogenesisand inflammation-driven arteriosclerosis in chronically-rejecting mousecardiac allografts. Permission for animal experimentation was obtainedfrom the State Provincial Office of Southern Finland. The mice and ratsreceived care in compliance with the “Guide for the Care and Use ofLaboratory Animals” prepared by the National Academy of Sciences andpublished by the National Academy Press (ISBN 0-309-05377-3, revised1996).

Rat and Mouse Heterotopic Heart Transplantation Model

Specific pathogen-free inbred male Dark Agouti (DA, RT1av1) and WistarFurth (WF, RT1u) rats (Scanbur, Sollentuna, Sweden) weighing 250-300 gand 2-3 months of age were used. Heterotopic cardiac allografts weretransplanted in abdominal position between fully MHC-mismatched strains.The donor heart was perfused through the inferior vena cava with 1 ml of+4° C. 0.9% NaCl with 500 IU heparin. The inferior and superior venacava, and pulmonary veins were ligated. The ascending aorta andpulmonary artery were excised distally, and the donor heart was removedand kept in +4° C. PBS. The allograft aorta and pulmonary artery werethen anastomozed to the abdominal aorta and vena cava inferior of therecipient. In the acute rejection model, no immunosuppression was usedand the syngrafts (DA->DA) and allografts (DA->WF) were harvested at 5days. In the chronic rejection model, the recipients receivedcyclosporine A (CsA, Novartis Basel, Switzerland) 2 mg/kg/d for thefirst week and 1 mg/kg/d thereafter, and the grafts were harvested at 8weeks. CsA was dissolved in Intralipid (100 mg/ml, Fresenius Kabi, BadHomburg, Germany) and was administered subcutaneously.

In the mouse model, specific pathogen-free inbred male BALB/c (B/c,H-2d) and C57BL/6J (B6, H-2b) mice (Harlan) weighing 25-30 g and 2-3months of age were used. The recipients received FK506 (intramuscularformulation, Astellas Pharma, Tokyo, Japan) subcutaneously 3.0 mg/kg/dfor the first week and 1.5 mg/kg/d thereafter as backgroundimmunosuppression, and the allografts were harvested at 8 weeks forhistological and immunohistochemical analysis. This immunosuppressionwas chosen after preliminary studies with different FK506 dosing, as thecurrent administration resulted in prolonged allograft survival anddevelopment of CAV.

Origin of Allograft Lymphatic EC

Transgenic marker gene mice that express LacZ under VEGFR-3 promoter(VEGFR-3/LacZ) were used to investigate the origin of allograft VEGFR-3lymphatic EC. To investigate recipient-derived VEGFR-3 expression in thetransplanted heart, Balb/c hearts were transplanted to VEGFR-3/LacZrecipients using the mouse chronic rejection heart transplant model.Replacement of allograft lymphatic EC with bone marrow (BM)-derivedcells was investigated using C57 mice that had received a BM transplantfrom GFP-expressing syngenic mice (GFP-BM) as Balb cardiac allograftrecipients using the mouse chronic rejection heterotopic hearttransplant model (n=3). Allografts were harvested at 8 weeks. Sampleswere first incubated in 2% paraformaldehyde for 30 min, then in 20%sucrose overnight, embedded in TissueTek and snap-frozen in liquidnitrogen.

Effect of VEGFR-3 Inhibition on Alloimmune Responses in Rat CardiacAllografts

To investigate the role of VEGFR-3 ligand inhibition in rat cardiacallografts, recipients were perfused in the beginning of the operationintraportally with adenoviral vectors (1×10⁹ pfu in 1 ml) encodingcontrol vector (Ad.LacZ or Ad.GFP) or soluble VEGFR-3 receptor(Ad.VEGFR-3-Ig). The recipients received CsA 1.0 mg/kg/d as backgroundimmunosuppression. The recipient livers and the transplanted allograftswere harvested on day 5 (ad.GFP. n=7; ad.VEGFR-3-Ig n=7) to investigatethe efficiency of adenoviral gene transfer, and alloimmune activation inthe allograft. The effect of intraportal ad.VEGFR3—Ig perfusion oncardiac allograft survival (ad.LacZ, n=10; ad.VEGFR-3-Ig, n=10) wasinvestigated by harvesting allografts at 8 weeks or if the graftfunction deteriorated. Hepatocyte GFP expression was detectedimmunohistochemically.

ELISA

ELISA (Quantikine-R&D Systems) was used to detect the presence ofVEGFR-3-Ig in rat serum collected at day 5 postransplantation,confirming the functionality of the adenoviral gene transfer in oursystem.

Effect of VEGFR-3 Inhibition in Chronically-Rejecting Mouse CardiacAllografts

To investigate the functional role of VEGFR-3, mouse cardiac allograftrecipients were treated with 800 μg of rat IgG (n=7; Sigma-Aldrich, St.Louise, Mo.) or rat anti-mouse VEGFR-3 neutralizing antibody (n=8;mF4-31C1, ImClone, New York, N.Y.). Antibodies were administeredintraperitoneally every third day for four weeks, starting immediatelyafter operation.

Histology

Cardiac transplant arteriosclerosis was determined by two independentobservers in blinded manner from paraformaldehyde-fixed paraffinsections stained with hematoxylin-eosin and Resorcin fuchsin forinternal elastic lamina using computer-assisted image processing(Axiovision 4.4, Carl Zeiss, Oberkochen, Germany) and measuring the areasurrounded by the internal elastic lamina and vessel lumen. Arterialocclusion percentage was determined as the ratio of neointimal area andinternal elastic lamina area.

Immunohistochemistry

Cryostat sections were stained using peroxidase ABC method (VectastainElite ABC Kit; Vector Laboratories, Burlingame, Calif.) and the reactionwas revealed by 3-amino-9-ethylcarbazole (AEC, Vectastain).Immunofluorescense double stainings were performed using a sequentialapproach and Alexa Fluor 488 (green) and Alexa Fluor 568 (red),(Promega, Madison, Wis.) secondary antibodies. Antibodies and dilutionsused were CD4 (5 μg/ml, 22021D), CD8 (5 μg/ml, 22071D), ED1 (5 μg/ml,22451D), CD111β (5 μg/ml, 553308) and IL-2Rα (5 μg/ml, 22090D) fromBDPharmingen, San Diego, Calif.; Ki67 (1:2000, NCL-Ki67p) fromNovocastra Laboratories Ltd, New Castle, UK; rabbit anti-mouse affinitypurified LYVE-1 (1:1000 with TSA amplification) and Anti-mouseCCL-21/6Ckine Antibody (1:200) from Professor Kari Alitalo; VEGF-C (0.5μg/ml, ab9546) and anti-GFP (1:200, ab 290) from Abcam, Cambridge, UK;mouse anti-rat OX-62 (10 μg/ml, MCA1029G), RECA-1 (50 μg/ml, MCA970) andmajor histocompatibility complex (MHC) class II (10 μg/ml, MCA46R) fromSerotec, Oxford, UK; VEGFR-3 (200 μg/ml, AF743) from R&D Systems,Minneapolis, Minn.; PROX-1 (0.01 mg/ml, DP 3501P) from Acris Antibodies,Hiddenhausen, Germany. All analyses were performed in a blinded mannerby two independent observers.

Analysis of the Immunohistochemical Stainings

Graft-infiltrating inflammatory cells and LYVE-1⁺, VEGFR-3⁺ or CCL-21⁺lymphatic vessels with clear lumen were counted from four random fieldsfrom each quadrant of the section's parenchyme with 40× magnificationand are given as the mean number of positive cells or vessels per mm2.Lymphatic vessels were also counted from the epicardium of the sectionand the amount of vessels are given as the mean number of positivevessels per mm².

RNA Isolation and Reverse Transcription

Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany)(n=4-6 per group). Reverse transcription of mRNA was carried out from100 ng total RNA in a final volume of 20 μl, using 200 U M-MLV reversetranscriptase (Sigma-Aldrich), with 20 U recombinant RNasin ribonucleaseinhibitor (Promega), 0.5 mM dNTPs (Sigma-Aldrich), and 2.5 μM randomnonamers (Sigma-Aldrich). After RT, 40 μl of nuclease-free water wasadded to each cDNA and 3 μl of each sample was used in each subsequentPCR reaction.

Real-Time PCR

External standards were used to generate a standard curve for each geneof interest. The templates of these standards consisted of PCR fragmentsgenerated with the same primers as used in real-time PCR. The DNAconcentrations were determined by spectrophotometry (Eppendorf, Hamburg,Germany), followed by calculation of the PCR fragment concentrations.For each standard curve, 10-fold serial dilutions were made startingfrom 10⁷ PCR fragments. The number of copies of the gene of interest wascalculated from the corresponding standard curve using LightCyclersoftware (Roche, Basel, Switzerland).

Real-time RT-PCR reactions were carried out in a LightCycler usingLightCycler FastStart DNA MasterPLUS SYBR Green I mix (Roche), primerconcentrations of 0.4 μM, and a cDNA amount corresponding to 5 ng totalRNA in a reaction volume of 10 μl. A typical protocol included a 10-mindenaturation step at +95° C. followed by 35 cycles with a +95° C.denaturation step for 10 sec, annealing at +59° C. for 10 sec, andextension at +72° C. depending on the length of the product (1 sec for25 bp). Measurement of the PCR product was performed at the end of eachextension period. Amplification specificity was checked using meltingcurve analysis. Results are given in relation to 18S rRNA moleculenumbers.

The following primers for rat IL-2 (Gene Bank accession no.NM_(—)053836), IL-4 (acc. No. NM_(—)201270), IL-6 (acc. No.NM_(—)012589), IL-10 (acc. No. NM_(—)012854), TNF-α (acc. No.NM_(—)012675), IFN-γ (acc. No. NM_(—)138880), CCL-21 (acc. No.NM_(—)011124) and FOXP-3 (acc. No. XM_(—)228771) were used:

IL-2 fwd 5′-CTGAGAGGGATCGATAATTACAAGA-3′; (SEQ ID NO: 129) bwd5′-ATTGGCACTCAAATTTGTTTTCAG-3′; (SEQ ID NO: 130) IL-4 fwd5′-ATGTTTGTACCAGACGTCCTTACG-3′; (SEQ ID NO: 131) bwd5′-TGCGAAGCACCCTGGAA-3′; (SEQ ID NO: 132) IL-6 fwd5′-CCCAACTTCCAATGCTCTCCTAATG-3′; (SEQ ID NO: 133) bwd5′-GCACACTAGGTTTGCCGAGTAGACC-3′; (SEQ ID NO: 134) IL-10 fwd5′-TAAGGGTTACTTGGGTTGCC-3′; (SEQ ID NO: 135) bwd5′-TATCCAGAGGGTCTTCAGC-3′; (SEQ ID NO: 136) TNF-α fwd5′-CTGTGCCTCAGCCTCTTCTCATTC-3′; (SEQ ID NO: 137) bwd5′-TTGGGAACTTCTCCTCCTTGTTGG-3′; (SEQ ID NO: 138) IFN-γ fwd5′-GAGGTGAACAACCCACAGA-3′; (SEQ ID NO: 139) bwd5′-TATTGGCACACTCTCTACCC-3′; (SEQ ID NO: 140) CCL-21 fwd5′-CCCTGGACCCAAGGCAGT-3′; (SEQ ID NO: 141) bwd5′-AGGCTTAGAGTGCTTCCGGG-3′; (SEQ ID NO: 142) and FOXP-3 fwd5′-GCTTGTTTGCTGTGCGGAGAC-3′; (SEQ ID NO: 143) bwd5′-GTTTCTGAAGTAGGCGAACAT-3′. (SEQ ID NO: 144)

Flow Cytometry

The cardiac allograft spleens were harvested at 5 days after thetransplantation to RPMI-1640 medium. The tissue was homogenized with ascalpel and 1×10⁶ spleen cells were incubated with FITC- orPE-conjugated antibodies for 15 minutes at room temperature. The cellswere then washed twice with PBS and analyzed with a FACScan (BectonDickinson) flow cytometer. Antibodies used were CD45-FITC (MCA43FT,Serotec), CD68-RPE (MCA341PE, Serotec), OX62-RPE (MCA1029PE, Serotec).IgG1-FITC (MCA43FT, Serotec) and IgG-RPE (MCA1209PE) were used asnegative isotype controls.

Statistics

All data are given as mean ±SEM and analyzed by parametric Student Ttest, or by log-rank test (graft survival) using SPSS for Windowsversion 11.5.1 (SPSS inc., Chicago, Ill.). P<0.05 was regarded asstatistically significant.

Results

Chronic Alloimmune Stimulus Induces Myocardial Lymphangiogenesis inCardiac Allografts

As lymphatic growth is often seen during inflammation, we evaluatedwhether acute or chronic rejection induces lymphangiogenesis in ratcardiac allografts by using lymphatic endothelium-specific hyaluronanacid receptor-1 (LYVE-1). In the acute rejection model, fullyMHC-mismatched rat heterotopic cardiac allograft recipients werenon-immunosuppressed, and allografts developed an intense acuterejection at 5 days. In the chronic rejection model, allograftrecipients received suboptimal cyclosporine A (CsA) immunosuppression,and allografts showed chronic rejection with moderate allograftinflammation, myocardial fibrosis, and arteriosclerosis at 8 weeks. Wefound small and large LYVE-1⁺ vessel structures in the myocardium ofnormal non-transplanted and transplanted hearts. These vessels opened tolarger epicardial collecting LYVE-1+ lymphatic vessels. In normal heartsand syngeneic controls, LYVE-1⁺ lymphatic vessel density was two timeshigher in the epicardial area than in the myocardium. Chronic rejectiondoubled the myocardial LYVE-1⁺ lymphatic vessel density, suggestingactive lymphangiogenesis during chronic allograft inflammation. Acuterejection decreased the epicardial lymphatic vessel density, possiblyindicating lymphatic vessel destruction during intense inflammation.

Immunofluorescence double stainings of chronically-rejecting cardiacallografts demonstrated the expression of lymphatic endothelial celltranscription factor Prox-1 in the nucleus of the LYVE-1⁺ cells,confirming the lymphatic phenotype. This was further supported by theobservation that LYVE-1 and rat vascular endothelial cell antigen-1(RECA-1) were not expressed in same vessels, suggesting that these aremarkers of lymphatic and vascular EC in the rat, respectively. Theproliferation marker Ki67 was infrequently found in LYVE-1⁺ EC, whereasseveral Ki67⁺ LYVE-1-allograft-infiltrating mononuclear cells weredetected outside LYVE-1⁺ lymphatic vessels. CD4⁺ and CD8⁺ T lymphocyteswere mainly detected outside the LYVE-1⁺ lymphatic vessels. In contrast,ED1⁺ macrophages and OX-62⁺ DC were found both outside and inside theLYVE-1⁺ vascular structures, indicating that the lymphatic vessels incardiac allografts are functional in transferring APC.

Macrophages and CD4⁺ Lymphocytes are the Major Source of VEGF-C inChronically-Rejecting Cardiac Allografts

Our finding that chronic alloimune stimulus induces lymphangiogenesis incardiac allografts prompted us to investigate the expression of a potentlymphangiogenic cytokine VEGF-C in transplanted hearts. VEGF-C wasmainly expressed in graft-infiltrating mononuclear cells. The density ofVEGF-C⁺ cells was similar in non-transplanted hearts, acutely rejectingcardiac allografts, and syngenic controls, whereas myocardialVEGF-C+density was two times higher in cardiac allografts undergoingchronic rejection. Immunofluorescense double stainings showed that asubset of ED1⁺ macrophages and CD4⁺ lymphocytes were VEGF-C positive,whereas CD8⁺ lymphocytes did not show VEGF-C immunoreactivity. Thesefindings indicate that during chronic cardiac allograft rejection,macrophages and CD4⁺ T lymphocytes are the major source forlymphangiogenic VEGF-C.

VEGFR-3 is Expressed in Lymphatic Endothelium and A Subset of DendriticCells in Cardiac Allografts

We determined the expression of VEGFR-3—the receptor for VEGF-C intransplanted rat hearts. VEGFR-3 immunoreactivity was detected inlymphatic-like vessels of non-transplanted and transplanted hearts.Mononuclear cells were encountered inside the VEGFR-3⁺ vessels ofcardiac allografts. Immunohistochemical staining of consecutive sectionsshowed that VEGFR-3 and CCL21 expression was localized in the samelymphatic EC of chronically rejecting allografts. The density ofVEGFR-3⁺ vessels was generally lower in the myocardium than in theepicardial area. Myocardial VEGFR-3⁺ vessel density was three timeshigher in chronically-rejecting cardiac allografts than in syngeniccontrols.

Immunofluorescence double stainings of chronically-rejecting cardiacallografts showed that endothelial VEGFR-3 immunoreactivity co-localizedwith LYVE-1 expression, although not all LYVE-1⁺ vessels expressedVEGFR-3. In addition to the lymphatic VEGFR-3 expression, we alsodetected lower VEGFR-3 immunoreactivity in occasionalallograft-infiltrating mononuclear cells. The majority of these VEGFR-3+cells were identified as OX-62⁺ DC, whereas very few ED1⁺ macrophages,and no CD4⁺ or CD8⁺ cells expressed VEGFR-3. ED1⁺ macrophages were oftenencountered inside the VEGFR-3⁺ lymphatic vessels. Collectively, thesefindings indicate that VEGFR-3 is expressed in lymphatic vessels andsubset of DC in cardiac allografts.

Cardiac allograft VEGFR-3+ lymphatic vessels but not spleen mariginalzone VEGFR-3⁺ vessels produce CCL21

Distinct molecular properties of lymphatic endothelial cells, such asproduction of CCL21 chemokine (Kriehuber et al., J. Exp. Med.,194:797-808, 2001), are essential in the specialized function oflymphatic vessels in transferring APCs to secondary lymphoid organs.This prompted us to investigate whether CCL21 is produced by cardiacallograft lymphatic vessels. Immunohistochemical staining of consecutivesections showed that VEGFR-3 and CCL21 were expressed in the samelymphatic vessels of chronically-rejecting allografts. In contrast,VEGFR-3 and CCL21 expression did not co-localize in normal spleen or inthe spleen of cardiac allograft recipients. Results indicated thatVEGFR-3 was expressed in the endothelium of vessel-like structuresaround spleen T cell zones, whereas CCL21 expression localized to whitepulp stromal cells and to the central arterioles. The finding thatallograft VEGFR-3+ lymphatic vessels produce CCL21 suggests thatlymphatic endothelial cell-derived chemokine-mediated signals arepresent at the exit of APCs from cardiac allografts similarly as incorneal allografts (Jin et al., Mol. Vis., 13:626-634, 2007). Incontrast, the VEGFR-3⁺ vessels in the spleen (presumably capillaries orvenous sinuses of the marginal zone) did not produce CCL21 but may beinvolved in leukocyte trafficking through non-CCL21-mediated mechanisms.

The Majority of VEGFR-3+ Lymphatic EC in Chronically-Rejecting MouseCardiac Allografts are Donor-Derived

Recent evidence suggests that BM-derived and non-BM-derived VEGFR-3⁺cells contribute to lymphangiogenesis (Kerjaschki et al., (2006),“Lymphatic endothelial progenitor cells contribute to de novolymphangiogenesis in human renal transplants,” Nat. Med. 12: 230-234;and Maruyama et al., (2005), “Inflammation-induced lymphangiogenesis inthe cornea arises from CD11b-positive macrophages,” J. Clin. Invest.,115: 2363-2372). As we found active lymphangiogenesis inchronically-rejecting cardiac allografts, we next used marker gene miceas cardiac allograft recipients to determine the origin of VEGFR-3⁺lymphatic EC. First, C57/bl mice that had received BM transplantationfrom GFP mice (GFP-BM) were used as heart transplant recipients allowingthe detection of BM-derived cells in cardiac allografts. The recipientswere treated with suboptimal FK506 immunosuppression to prevent severeacute rejection, and the cardiac allografts were harvested eight weeksafter the transplantation. Immunofluorescence double stainings showedthat BM-derived GFP⁺ cells localized mainly around VEGFR-3⁺ lymphaticvessels. Less than 4% of the BM-derived GFP⁺ cells co-localized withVEGFR-3⁺ lymphatic EC.

As some of these GFP⁺ cells may actually be BM-derived inflammatorycells migrating to the VEGFR-3⁺ lymphatic vessels, Balb/c hearts werenext transplanted to mice that express LacZ under VEGFR-3 promoter(VEGFR-3/LacZ, C57/bl background, n=3), to allow the direct detection ofrecipient-derived VEGFR-3⁺ lymphatic cells in the allografts. The x-galstaining revealed epicardial lymphatic endothelial VEGFR-3 expression inthe VEGFR-3/LacZ recipient's own heart. In contrast, norecipient-derived VEGFR-3⁺ lymphatic EC were encountered in themyocardium of wild type cardiac allografts transplanted to VEGFR-3/LacZrecipients. These results indicate that the replacement of cardiacallograft VEGFR-3⁺ lymphatic EC with recipient BM-derived, ornon-BM-derived cells is rare in this chronic rejection heterotopic hearttransplantation model.

VEGFR-3 Inhibition Improves Cardiac Allograft Survival

We next determined the effect of VEGFR-3 inhibition on alloimmuneresponse by injecting suboptimally immunosuppressed rat cardiacallograft recipients intraportally with adenovirus vector encoding thesoluble form of VEGFR-3 (Ad.VEGFR-3-Ig, VEGF-C/D-trap). Hepatocyte GFPexpression was seen in Ad.GFP-perfused recipients, but not inAd.VEGFR-3-Ig-perfused recipients. See FIGS. 1A and 1B. Also, the serumlevels of VEGFR-3-Ig were elevated in the Ad.VEGFR-3-Ig-perfused animals5 days after transplantation (FIG. 1C), and remained elevated for atleast 21 days, confirming the functionality of the adenoviral genetransfer.

Next, we investigated the effect of VEGFR-3 inhibition on the survivalof fully MHC-mismatched rat cardiac allografts. In non-immunosuppressedcardiac allograft recipients, intraportal Ad.VEGFR-3-Ig-perfusionincreased allograft survival from 4.9 to 6.0 days (p<0.05, n=7 pergroup). Intraportal Ad.VEGFR-3-Ig-perfusion in cardiac allograftrecipients receiving suboptimal dose of CsA significantly improvedallograft survival. See FIG. 1D. Our results thus suggest that VEGFR-3inhibition together with CsA background immunosuppression markedlyprolongs long-term allograft survival while it only has a marginaleffect as sole treatment in a fully MHC-mismatched model.

VEGFR-3 Inhibition Decreases Cardiac Intragraft CCL21 Production,Alloimmune Activation and Effector Cell Recruitment

To clarify the underlying mechanisms behind the beneficial effect onallograft survival, we used the same experimental setting (Ad.VEGFR-3perfusion and suboptimal CsA immunosuppression) and harvested theallografts 5 days after transplantation. Ad.VEGFR-3-Ig-perfusiondecreased the density of, graft infiltrating CD8⁺ T cells, andalloimmune activation in the form of IL-2Rα⁺ and MHC class IIexpression. In contrast, no changes in graft infiltrating ED1⁺macrophages, allograft CD4⁺ cells or OX-62⁺ DC s or the density ofLYVE-1⁺ vessels were observed.

Real time RT-PCR showed that intraportal Ad.VEGFR-3-Ig perfusionresulted in a two-fold decrease in allograft CCL21 mRNA expression. Nosignificant changes were observed in IL-6, Foxp3, INF-γ, IL-10, TNF-α,NFκβ, lymphotoxin (LT)-α or LT-β mRNA levels. Together, these findingsindicate that VEGFR-3 inhibition decreases alloimmune activation andinfiltration of effector cells in cardiac allografts together with adecrease in intragraft CCL21 production.

VEGFR-3 Inhibition Decreases Dendritic Cell Recruitment to Spleen

We next investigated whether the reduction of alloimmune response withVEGFR-3 inhibition after heart transplantation was associated withimpaired APC recruitment to recipient secondary lymphoid organs usingFACS analysis from peripheral blood and spleen leukocytes. Ad.VEGFR-3-Igperfusion decreased the proportion of OX-62⁺ DC, and ED1⁺ cells (p=NS)of spleen CD45⁺ leukocytes. The proportion of OX-62⁺ cells of peripheralblood leukocytes was 12.7±2.5% in Ad.GFP group and 15.7±4.2% inAd.VEGFR-3 group. This indicates that the decrease in spleen DC was dueto impaired APC homing to the spleen rather than impaired APCmobilization from the BM. Results further indicated that VEGFR-3-Igincreased the proportion of VEGFR-3⁺ leukocytes in peripheral bloodpossibly due to a trapping effect. VEGFR-3-Ig did not change theproportion of OX-62⁺ DC in peripheral blood but decreased therecruitment of OX-62⁺ DC to the recipient spleen. These results indicatethat VEGFR-3 regulates APC traffic to secondary lymphoid organs.

VEGFR-3 Inhibition Regulates Chemokine Balance Between Allograft andSecondary Lymphoid Organs

RT-PCR analysis of a subpopulation (n=3) revealed that VEGFR-3inhibition was associated with over two-fold increase in spleen mRNAlevels of CCL21, IL-10, and Foxp3. Spleen VEGFR-3 immunoreactivity wasmainly detected in lymphatic-like vessel structures surrounding the Tcell zones, whereas CCL21 immunoreactivity was mainly detected in the Tcell zones and the central arterioles, and did not co-localize withVEGFR-3⁺ vessels. In cardiac allografts on the other hand, CCL21 wasmainly expressed in VEGFR-3⁺ lymphatic-like vessels. These observations,together with the unexpected CCL21 response after VEGFR-3 inhibition,may indicate that CCL21 is differentially regulated in peripheraltissues and secondary lymphatic tissue.

We performed real time RT-PCR analysis of the recipient spleen 5 daysafter heart transplantation to investigate whether VEGFR-3-Ig decreasedCCL21 production in the spleen similarly as in the allograft. Incontrast to the results in the transplanted heart, treatment withVEGFR-3-Ig actually resulted in 1.5 times higher CCL21 mRNA levels inthe spleen. We also observed that VEGFR-3-Ig markedly increased theratio of spleen-to-allograft CCL21 mRNA from 9:1 to 23:1. Thedifferential effect of VEGFR-3 inhibition on allograft and spleen CCL21mRNA production may be explained by the finding that cardiac allograftVEGFR-3+ lymphatic vessels produced CCL21 whereas VEGFR-3 and CCL21 werenot produced by the same cells in the spleen. Further RT-PCR analysis ofthe spleen revealed that VEGFR-3 inhibition resulted in a significantincrease in Treg transcription factor Foxp3. This is interesting in thelight of recent reports that CCL21 plays a vital role in homing,localization and function of Tregs (Schneider et al., J. Exp. Med.,204:735-745, 2007; Kocks et al., J. Exp. Med., 204:723-734, 2007). Inaddition, VEGFR-3 inhibition did not significantly alter spleen mRNAlevels of IL-10 (Hori et al., Science, 299:1057-1061, 2003), IL-6,IFN-γ, TNF-

, NF-κB, LT-

, and LT-β at 5 days after transplantation. These results suggest thatVEGFR-3 inhibition regulates chemokine balance between allograft andsecondary lymphoid organs in favour of attenuated immune response.

VEGFR-3 Neutralizing Monoclonal Antibody Decreases Inflammation andInflammation-Driven Arteriosclerosis in Chronically-Rejecting MouseCardiac Allografts

We wanted to confirm the results of VEGFR-3 inhibition with neutralizingantibodies against VEGFR-3 in another chronic rejection hearttransplantation model. Mouse cardiac allograft recipients receivedsuboptimal FK506 immunosuppression to prevent intense acute rejectionand to allow the development of chronic rejection at 8 weeks. Inaddition, the recipients were treated either with rat IgG or ratanti-mouse neutralizing antibodies (VEGFR-3 mAb, ImClone) againstVEGFR-3 for one month. At two months, the effect of VEGFR-3 inhibitionreduced the density of VEGFR-3⁺ and CCL-21⁺ lymphatic vessels in theallograft. In addition, VEGFR-3 inhibition reduced the density of CD4⁺lymphocytes, CD8⁺ lymphocytes, and CD11b⁺ myelomonocytic cells.Interestingly, treatment with VEGFR-3 mAb significantly decreased themean arterial occlusion compared to the control group. Our results thusshow that early treatment with neutralizing VEGFR-3 antibody decreasesallograft CCL21 production, inflammation, and arteriosclerosis inchronically-rejecting mouse cardiac allografts.

Finally, because lymphoid neogenesis (i.e., organization of chronicinflammatory infiltrates into functional ectopic germinal centers ortertiary lymphoid organs (TLOs)), has been linked with the formation ofchronic rejection (Thaunat et al., Proc. Natl. Acad. Sci. USA,102:14723-14728, 2005; Baddoura et al., Am. J. Transplant, 5:510-516,2005; Nasr et al., Am. J. Transplant., 7:1071-1079, 2007), weinvestigated the presence of TLOs in our chronically-rejecting cardiacallografts. Immunohistochemical stainings revealed a characteristicpattern of peripheral node adressin-positive high endothelial venules,and discrete B and T cell accumulation only in one allograft in thecontrol group (FIG. 10, A-C). This implies that lymphoid neogenesis isnot a critical phenomenon at least in the two month end-point of ourchronic rejection model, and the effect of VEGFR-3 inhibition on TLOformation thus cannot be evaluated in this model within this timeframe.

Analysis

The lymphatic network is adapted at both structural and molecular levelto transfer leukocytes out of tissues. The thin-walled lymphaticcapillaries provide easy access for interstitial cells and fluid,whereas the smooth muscle coverage and valves of the collectinglymphatic vessels provide unilateral movement towards secondary lymphoidorgans. Lymphatic EC have distinct molecular properties that reflecttheir function and have been utilized for the detection of lymphaticvessels. These cells express podoplanin, hyaluronan receptor LYVE-1,VEGFR-3 and inflammatory cytokine CCL21 that are not found in vascularEC. The identification of signals that regulate lymphatic growth—mostimportantly VEGF-C/VEGFR-3—has greatly improved our knowledge oflymphatic vessels in both physiological and pathological situations.

Effective transfer of APC from transplanted organs to secondary lymphoidorgans is critical for the priming of alloreactive T cells and thedevelopment of alloimmune responses that may be detrimental for theheart transplant recipient. In the current study, we found that chroniccardiac allograft rejection increased myocardial lymphatic vesseldensity. The capillary lymphatics in cardiac allografts opened toepicardial collecting lymphatic vessels, and expressed the lymphatictranscription factor Prox-1, LYVE-1, VEGFR-3, and CCL21. In addition,APC such as macrophages and DC were often encountered inside thesevessels, indicating vessel functionality. Active lymphangiogenesis isseen in human kidney transplants with nodular inflammatory infiltrates(Kerjaschki et al., (2004), “Lymphatic neoangiogenesis in human kidneytransplants is associated with immunologically active lymphocyticinfiltrates,” J. Am. Soc. Nephrol., 15: 603-612) and in otherinflammatory conditions. As the observed lymphangiogenesis inchronically-rejecting allografts in this study was accompanied with anincrease in VEGF-C-producing macrophages and CD4⁺ lymphocytes, ourresults suggest interplay of chronic inflammation, VEGF-C/VEGFR-3signalling, and lymphangiogenesis in cardiac allografts.

Lymphatic EC in the transplanted heart may originate from recipient BMcells, from recipient non-BM cells or from donor cells. Recently, it wasshown that recipient-derived lymphatic endothelial progenitorcells—possibly in the form of macrophages—participate inlymphangiogenesis of human kidney allografts (Kerjaschki et al., (2006),“Lymphatic endothelial progenitor cells contribute to de novolymphangiogenesis in human renal transplants,” Nat. Med. 12: 230-234).Also, macrophages may directly trans-differentiate to lymphatic EC inthe inflamed cornea (Maruyama et al., (2005), “Inflammation-inducedlymphangiogenesis in the cornea arises from CD11b-positive macrophages,”J. Clin. Invest. 115: 2363-2372), and may provide cytokines for theexpansion of resident lymphatics (Kerjaschki, D. (2005), “The crucialrole of macrophages in lymphangiogenesis,” J. Clin. Invest. 115:2316-2319). Here, we used marker gene mice as cardiac allograftrecipients and found that recipient-derived cells contribute onlyminimally to the formation of VEGFR-3+ lymphatic vessels in theheterotopically transplanted hearts. As Kerjascki et al found that about13% of Prox-1+ lymphatic vessels in rejected and nephrectomized kidneytransplants originated from the recipient, it is possible that theinvolvement of recipient-derived lymphatic progenitors is dependent onthe severity of allograft injury. Paralleling this hypothesis, thedegree of allograft injury may determine whether allograft vascular ECoriginate from the recipient—as in aortic transplantation—or from thedonor—as in cardiac allografts (Hillebrands et al., (2001), “Origin ofneointimal endothelium and alpha-actin-positive smooth muscle cells intransplant arteriosclerosis, J. Clin. Invest., 107: 1411-1422). Also,the actual effect of lymphatic endothelium chimerism of transplantedorgans on alloimmune responses remains unknown.

Chen et al (2004) have recently reported that VEGFR-3 inhibition impairsDC migration to draining LN and improves the survival of corneatransplants. These effects were possibly mediated through directinhibition of VEGFR-3+ DC migration independent of lymphangiogeniceffects. However, the functional role of VEGFR-3 after solid organtransplantation has been unclear. Here, systemic VEGFR-3 inhibitionusing adenoviruses encoding soluble VEGFR-3-Ig that traps VEGFR-3ligands decreased DC migration to spleen, alloimmune activation andimproved the long term survival of cardiac allografts. Similarly,treatment with neutralizing VEGFR-3 antibodies decreased allograftinflammation and development of inflammation-driven arteriosclerosis inthe chronically rejecting mice cardiac allografts. As we found VEGFR-3⁺DC in cardiac allografts, it is possible that VEGFR-3 inhibition haddirect effects on DC migration in the current study similar to thefindings in the corneal transplantation model (18).

In addition to the direct effects on VEGFR-3+ DC, our results suggestthat VEGFR-3 inhibition also had lymphatic EC— and chemokine-mediatedeffects. Specifically, both VEGFR-3 and CCL21, a chemokine for CCR7+APC, were co-expressed in allograft lymphatic EC, and VEGFR-3 inhibitiondecreased allograft CCL21 production. Our results thus indicate thatVEGFR-3 in allograft lymphatic EC may regulate the production of CCL21,that may in turn facilitate the movement of APC from the allograft tosecondary lymphoid tissue and subsequent alloimmune activation.Surprisingly, in contrast to the allograft, VEGFR-3 inhibition increasedCCL21 production in the spleen. VEGFR-3 was mainly expressed around thespleen T cell zones whereas the central arterioles and stromal cells ofthe T cell zones were the main source of CCL21 in the spleen. Therefore,the regulation of CCL21 production may be different in the heart and thespleen due to the differential pattern of VEGFR-3 and CCL21 expression.

In experimental studies, the survival of cardiac allografts is modestlyincreased in CCR7-deficient recipients as well as in CCL21-deficientrecipients (Forster et al., (1999), “CCR7 coordinates the primary immuneresponse by establishing functional microenvironments in secondarylymphoid organs,” Cell, 99: 23-33; and Colvin et al., (2005), “CXCL9antagonism further extends prolonged cardiac allograft survival inCCL19/CCL21-deficient mice,” Am. J. Transplant., 5: 2104-2113).Collectively, these studies show an important but not critical role ofCCL21/CCR7-signalling in alloimmune reactions.

In the present study, VEGFR-3 inhibition that resulted in decreasedCCL21 production did not completely prevent alloimmune responses inrecipients receiving suboptimal dose of CsA. Therefore, VEGFR-3inhibition alone may not completely prevent alloimmune responses intransplant recipients. For optimal results VEGFR-3 inhibition can beused as induction or adjuvant therapy in the prevention and treatment ofacute rejection, in addition to (in combination with) conventionalT-cell-targeted immunosuppression. Combination therapy targeting otherVEGFR's or PDGFR's or their cognate growth factors also is contemplated.Interestingly, VEGFR-3 inhibition resulted in over two-fold increase inspleen Foxp3 and IL-10 mRNA production. Therefore, VEGFR-3 inhibitionmay also have beneficial effects on Treg, but further studies are neededto clarify the effect of VEGFR-3 signalling on Tregs.

In conclusion, these results indicate that VEGF-C/VEGFR-3 signalling hasimportant effects on proximal events in cardiac allograft alloimmunityand inflammation-driven arteriosclerosis, possibly through regulatinglymphatic endothelial cell CCL21 production and leukocyte traffickingand through direct effects on VEGFR-3⁺ DC. As an important safetyaspect, adult lymphatic vessels are fairly resistant to VEGFR-3inhibition, suggesting that this treatment in transplant recipientswould also primarily inhibit lymphangiogenesis and the functionality oflymphatic vessels in contrast to regression of the existing lymphaticnetwork. Therefore, VEGFR-3 inhibition could be used as a non-Tcell-targeted induction therapy to regulate alloimmune activation aftersolid organ transplantation.

Example 8 VEGFR-1 and -2 Regulate Inflammation, Myocardial Angiogenesisand Arteriosclerosis in Chronically Rejecting Cardiac Allografts/R1

We investigated how the two vascular endothelial growth factor receptorsVEGFR-1 and VEGFR-2 regulate inflammation and angiogenesis inchronically rejecting cardiac allografts. As described below in detail,chronic rejection in mouse cardiac allografts induced primitivemyocardial, adventitial, and intimal angiogenesis with endothelialexpression of CD31, stem cell marker c-kit, and VEGFR-2. Experimentsusing marker gene mice or rats as cardiac allograft recipients revealedthat replacement of cardiac allograft endothelial cells with recipientbone-marrow- or non-bone-marrow-derived cells was rare and restrictedonly to sites with severe injury. Targeting VEGFR-1 with neutralizingantibodies in mice reduced allograft CD11b+myelomonocyte infiltrationand allograft arteriosclerosis. VEGFR-2 inhibition prevented myocardialc-kit+ and CD31+ angiogenesis in the allograft, and decreased allograftinflammation and arteriosclerosis. These results indicate an interplayof inflammation, primitive donor-derived myocardial angiogenesis, andarteriosclerosis in transplanted hearts, and further indicate thattargeting VEGFR-1 and -2 with inhibitors differentially regulate thesepathological reparative processes.

Materials Design

Mouse chronic rejection heart transplantation model andimmunohistochemical stainings were used to identify angiogenesis andprogenitor cells in allografts. Marker gene mice and rats, andstrain-specific major histocompatibility complex (MHC) class Iantibodies, were used to determine whether allograft EC originate fromthe donor, or from the recipient. Neutralizing antibodies were used toinvestigate the functional role of VEGFR-1 and VEGFR-2 on mouse cardiacallograft angiogenesis, inflammation, and arteriosclerosis.

Mouse Chronic Rejection Heterotopic Heart Transplantation Model

Heterotopic cardiac allografts were transplanted in abdominal positionfrom Balb (B/c, H-2^(d)) to C57 (B6, H-2^(b)) mice (Harlan, Horst, TheNetherlands). The recipients received sub-optimal FK506immunosuppression (i.m. formulation, Astellas Pharma, Tokyo, Japan) andthe allografts were harvested at 8 weeks.

Origin of Allograft Endothelial Cells

Tie1l/LacZ rats32 were used as allograft recipients (n=4) or donors(n=14, with or without immunosuppression) to investigate Tie1expression, and the origin of Tie1-positive EC in transplanted hearts.Contribution of BM-derived cells in allograft angiogenesis wasinvestigated using recipient mice with green fluorescentprotein-expressing BM cells (GFP-BM, n=3). See Rajantie et al., “Adultbone marrow-derived cells recruited during angiogenesis compriseprecursors for periendothelial vascular mural cells,” Blood, (2004);104: 2084-2086.

VEGFR-1 and VEGFR-2 Inhibition

Cardiac allograft recipients were treated with 800 μg of rat IgG (n=8;Sigma-Aldrich, St. Louis, Mo.), anti-VEGFR-1 antibody (n=9; MF1,ImClone, New York, N.Y.), anti-VEGFR-2 antibody (n=9; DC101, ImClone) ortheir combination (n=10) every third day for 10 doses, startingimmediately after the transplantation.

Histology and Immunohistochemistry

Arterial occlusion percentage was determined using morphometry.Immunohistochemical stainings were performed using peroxidase ABC methodor Alexa Fluor 488 (green) and 568 (red, Promega, Madison, Wis.)secondary antibodies.

Analysis of Immunohistochemical Stainings

Allograft parenchymal inflammatory cells and c-kit+capillaries werecounted from 16 random sections, and are summarized as the mean densityof positive cells or vessels. CD31 and α-SMA immunofluorescensestainings were analyzed with Axioplan 2 microscope and Axiovision 4.2analysis software (Carl Zeiss, Oberkochen, Germany) using asemiautomated script.

Real Time RT-PCR

Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany)(n=4-6 per group). RT-PCR reactions were carried out using LightCycler(Roche, Basel, Switzerland) and the results are given in relation to 18SrRNA molecule numbers.

Statistical Analysis

Data are mean ±SEM and analyzed by parametric ANOVA with Dunnett'scorrection to compare the treatment groups to the control group. Linearregression analysis was applied to evaluate relation of c-kit+ cells toCD11b+ cells and to cardiac allograft vasculopathy (CAV). P<0.05 wasregarded as statistically significant.

Results

Chronic Rejection Induces Primitive Myocardial Angiogenesis in CardiacAllografts

We detected only occasional stem cell marker c-kit immunoreactive cellsin cross-sections of non-transplanted mouse hearts. In contrast,numerous myocardial capillary-like c-kit+ cells and c-kit+ vein EC wereobserved in chronically-rejecting cardiac allografts harvested 2 monthsafter the transplant operation. In allografts with severearteriosclerotic changes, c-kit+ cells were also found in the adventitiaand intima of coronary arteries.

Allograft myocardial c-kit+ cells were nearly all positive forendothelial marker CD31, and co-expressed VEGFR-2. The majority ofc-kit+capillaries did not express proliferation marker Ki67, but somec-kit+ cells with nuclear Ki67 immunoreactivity were also detected.

In contrast to the preferential expression of VEGFR-2 in theendothelium, VEGFR-1 was mainly expressed in allograft α-SMA+ SMC. Inperipheral blood, over 50% of VEGFR-1⁺ cells coexpressed themyelomonocyte marker CD 11b. No specific immunoreactivity with IgGcontrol was observed.

A positive correlation was verified between the density ofc-kit+capillaries in the myocardium and the number ofallograft-infiltrating CD11b+myelomonocytic inflammatory cells, as wellas with the incidence of arteriosclerotic changes and the mean occlusionof allograft arteries. These results indicate that chronic rejection intransplanted hearts induces myocardial, adventitial and intimalangiogenesis with endothelial expression of primitive markers c-kit andVEGFR-2.

Endothelial Replacement with Recipient-derived Cells is Rare in CardiacAllografts

Because recipient-derived circulating EPC could differentiate to EC inthe transplanted heart, we determined the origin of cardiac allograft ECby using marker gene rats (Tie1/LacZ) or mice (GFP-BM) as allograftrecipients.

When Tie1/LacZ allografts were transplanted to wild type (WT)recipients, areas with abundant X-gal reactivity in venous and arterialallograft endothelium was detected, indicating Tie1 expression in thedonor EC.

Next, WT cardiac allografts were transplanted to Tie1/LacZ recipients todetect recipient-derived EC in the transplanted hearts. Only fewdonor-derived X-gal+EC, localizing to severely fibrotic areas, were seenin cross-sections in a total of 14 WT cardiac allografts.

Additionally, GFP-BM mice were used as cardiac allograft recipients,allowing the detection of BM-derived cells in the allografts. Themajority of allograft-infiltrating CD11b+myelomonocytic cells expressedGFP. Although GFP+ cells often surrounded allograft blood vessels, noco-localization with allograft CD31+ or c-kit+ capillaries was detected.

Donor- and recipient-specific MHC class I antibodies were used toidentify the source of EC in allograft arteriosclerotic arteries.Numerous recipient MHC class I+ cells were found around occludedarteries, whereas only few positive cells were detected in the intima.In contrast, abundant donor MHC Class I immunoreactivity was found inthe neointima. The contribution of recipient-derived SMC to neointimalformation was not assessed, as MHC Class I expression was low in SMC34.

VEGFR-2 Inhibition Normalizes C-kit+ and CD31+Capillary Density inChronically Rejecting Cardiac Allografts

To investigate the functional role of VEGFR-1 and -2, chronicallyrejecting mouse cardiac allograft recipients with suboptimal FK506immunosuppression were treated with rat IgG (n=8); or with antibodiesagainst VEGFR-1 (MF1, n=9), antibodies against VEGFR-2 (DC 101, n=9), orboth antibodies against VEGFR-1 and R-2 (n=10) for thirty days. Twomonths after heart transplantation, the antibodies targeting VEGFR-2reduced the density of myocardial c-kit+capillaries and CD31+capillaries in the allograft to the level found in non-transplantedmouse hearts. VEGFR-1 inhibition also resulted in a smaller decrease inc-kit+capillary density (p=NS with Dunnett's correction, p<0.05 with LSDcorrection). VEGFR-1 or -2 inhibition did not change the density of SMCcoated vessels (α-SMA+), indicating that VEGFR-2 inhibition specificallyregulated angiogenesis at microvascular level.

VEGFR-1 and -2 Inhibition Reduces Inflammation in Chronically RejectingCardiac Allografts

Immunohistochemical analysis showed that targeting VEGFR-1, VEGFR-2, orboth profoundly reduced the density of allograft-infiltratingCD11b+myelomonocytic cells. VEGFR-2 inhibition also resulted in asimilar reduction in CD8⁺ and CD4⁺ lymphocyte density in the allograft(for the combination group: p=NS with Dunnett's correction and p<0.05with LSD correction).

VEGFR-1 and -2 Inhibition Reduces Arteriosclerosis in ChronicallyRejecting Cardiac Allografts

Morphometrical analysis of allograft arteries revealed that targetingVEGFR-1, VEGFR-2, or both decreased the incidence of allograft arterieswith intimal changes from about 55% in the IgG control mice to under 40%in the mice receiving anti-VEGFR-1 (p<0.05), anti-VEGFR-2 (p<0.05), orboth (p<0.01). A similar result was also obtained on the mean occlusionof allograft arteries (about 18% arterial occlusion in the IgG controls,compared to about 7% in the anti-VEGFR-1 mice (p<0.01), about 11% in theanti-VEGFR-2 mice (p<0.05), and about 10% in the mice receiving bothantibodies (p<0.05). These results indicate that both VEGFR-1 and -2 areinvolved in events leading to CAV.

Effect of VEGFR-1 and -2 Inhibition on Allograft Cytokine mRNA Levels

Finally, we used real time RT-PCR to determine the mRNA levels ofinflammatory cytokines IFN-inducible protein-10 (IP-10) and monocytechemotactic protein-1 (MCP-1) that are potentially regulated by VEGF incardiac allografts, and the mRNA levels of stem cell factor (SCF) thatis the ligand for c-kit. The analysis revealed that VEGFR-2 inhibitiondecreased allograft IP-10 mRNA by approximately 50% alone, and by 75% incombination with VEGFR-1 inhibition, and MCP-1 mRNA by 50%. In contrast,allograft TNF-α and SCF mRNA levels were similar in the control andtreatment groups. These results indicate that the VEGFR-2 inhibitionregulated at least in part the T cell and monocyte recruitment bydecreasing IP-10 and MCP-1 production, respectively. Also, the effect ofVEGFR-2-inhibition on c-kit+ capillaries was not associated with changesin SCF production.

Analysis

Angiogenesis is a prominent feature in the intima and adventitia ofcardiac allograft coronary arteries and it may be a driving force forthe development of CAV. The results of these experiments demonstratethat, in addition to intimal and adventitial angiogenesis, chronicrejection induces the expression of primitive markers c-kit and VEGFR-2in allograft myocardial capillaries. As the density of myocardialc-kit+capillaries correlated with the severity of cardiac allograftinflammation and arteriosclerosis, alloimmune and ischemic stimuli maybe important regulators of the myocardial angiogenesis we observed. Thisprimitive ckit+ angiogenic response probably represents a repair processthat, interestingly, in light of the present VEGF intervention results,may in fact aggravate inflammation and arteriosclerosis in transplantedhearts. Importantly, there may be a balance between early capillaryformation and later destruction of allograft capillaries as seen in skintransplants. (See Moulton et al., “Angiogenesis in the huPBL-SCID modelof human transplant rejection,” Transplantation, (1999); 67:1626-1631.)

In experiments using marker gene animals and donor- orrecipient-specific antibodies, we found only few recipient-derived EC inthe transplanted hearts and they were restricted to severely fibroticareas. These observations suggest that recipient-derived circulatingcells do not differentiate into allograft EC unless the injury to theallograft extensive. Although this notion argues against directinvolvement of recipient-derived EPC in allograft angiogenesis, thesecirculating cells may have important paracrine effects. Our results onthe origin and c-kit+ phenotype of allograft EC further indicates thatdonor-derived progenitor cells—such as resident cardiac stem cells oradventitial stem cells—directly participate in allograft angiogenesis.Alternatively, the hypoxic and inflammatory signals related to thetransplantation may have induced dedifferentiation of allograft EC to amore primitive phenotype. Interestingly, EPC-derived soluble factorssuch as VEGF, VEGF-B, stromal cell derived factor-1, and insulin-likegrowth factor-1, and also hepatocyte growth factor may regulate thefunctions of c-kit+cardiac progenitor cells, and the present resultssuggest important role for VEGFR-2.

VEGF is perhaps the most important angiogenic cytokine and it also hasmany proinflammatory properties. The present findings support the theoryof regulatory role of VEGF in the pathogenesis of alloimmune responsesand CAV in transplanted hearts, and shed light to the mechanisms, andthe two VEGFR involved. In transplanted hearts VEGFR-2 inhibitionreduced myocardial angiogenesis to the level seen in normal hearts,consistent with the important angiogenic role for VEGFR-2. In addition,targeting VEGFR-2 decreased inflammatory cell infiltration, andproduction of IP-10 and MCP-1 in the allograft, similarly to previousreports with anti-VEGF therapies. (See, e.g., Reinders et al.,“Proinflammatory functions of vascular endothelial growth factor inalloimmunity,” J. Clin. Invest., (2003); 112:1655-1665.) Our resultsthus suggest that VEGFR-2 in cardiac allografts functions mainly at theendothelial level and regulates both pathological capillary angiogenesisand inflammation. Involvement of VEGFR-2 in cardiac inflammation may bea more general phenomenon, as the receptor participates in cardiacdysfunction during sepsis, and also in vascular permeability followingmyocardial infarction.

In contrast to VEGFR-2, VEGFR-1 was primarily found in allograft SMC andin peripheral blood myelomonocytic cells. As VEGFR-1 directly regulatesSMC during arterial injury, VEGFR-1 inhibition in the current study mayhave directly decreased SMC recruitment to the intima. VEGFR-1inhibition also profoundly reduced myelomonocyte recruitment to theallograft, consistent with its role in monocytes and inflammatorydiseases. Although VEGFR-2 inhibition prominently decreased the densityof myocardial c-kit+ cells, VEGFR-1 inhibition had a similar but moresubtle effect. This indicates that also VEGFR-1 may in part regulate thecapillary angiogenesis, and possibly involves cross-talk with VEGFR-2,or in-direct inflammation-mediated effects. The reason why combinedVEGFR-1 and -2 inhibition did not have a beneficial additive effect maybe explained by the moderate injury in the current experimental setting.Supporting this, our unpublished trachea transplantation findings showadditive beneficial effect after severe but not after moderate trachealinjury. (See also Sho et al., “Function of the Vascular EndothelialGrowth Factor Receptors Flt-1 and Flk-1/KDR in the Alloimmune ResponseIn Vivo,” Transplantation, (2005); 80: 717-722.)

In summary, these experiments demonstrated that chronic rejection incardiac allografts induced donor-derived capillary angiogenesis. Also,selective VEGFR-inhibition prevented allograft angiogenesis and hadbeneficial effects on inflammation and arteriosclerosis. These resultsindicate therapeutic applications for anti-VEGF strategies duringpathological angiogenesis and inflammation in transplanted hearts.

Example 9 Combination Therapy Targeting Receptors of Multiple GrowthFactors

The data in Examples 7 and 8 implicate VEGFR-1, VEGFR-2, and VEGFR-3(and the growth factor ligands of these receptors) in chronic allograftrejection, particularly with reference to the model system used: cardiactransplants.

The experiments of Examples 7 and 8 are modified in that newcombinations of inhibitors of growth factors and/or growth factorreceptors are employed, and the protective effects of the combinationsare evaluated. Evidence exists that the PDGF receptors and PDGF ligandsmay have a role in allograft disease. See, e.g., Nykanen et al.,“Angiogenic Growth Factors in Cardiac Allograft Rejection,”Transplantation, (2006); 82: S22-S24, incorporated herein by reference.It is expected that combinations of inhibitors that are directed toreceptors of distinct growth factor ligands (and/or directed to distinctgrowth factors themselves) will have additive or synergistic effects.All combinations described herein are specifically contemplated,including but not limited to the following:

(a) inhibitor of VEGFR-3 interaction with its ligands (VEGF-C or D), incombination with one or more inhibitors of VEGFR-1 and its ligands, orinhibitors of VEGFR-2 and its ligands, or both;

(b) inhibitor of VEGFR-3 interaction with its ligands, in combinationwith one or more inhibitors of PDGFR-alpha and its ligands, orPDGFR-beta and its ligands, or both;

(c) inhibitor of VEGFR-3 interaction with its ligands in combinationwith both (i) inhibitor of VEGFR-1 and its ligands, or inhibitor ofVEGFR-2 and its ligands; and (ii) inhibitor of PDGFR-alpha and itsligands, or PDGFR-beta and its ligands;

(d) inhibitors of VEGFR-3, VEGFR-2, VEGFR-1, PDGFR-alpha, and PDGFR-betawith their respective ligands;

(e) inhibitors of VEGFR-1 or VEGFR-2 in combination with inhibitors ofPDGFR-alpha or PDGFR-beta, and their respective ligands.

The inhibition of multiple receptors or ligands can be achieved withmultivalent inhibitor substances described herein; small moleculenon-specific inhibitors (e.g., tyrosine kinase inhibitors); or withco-administration of multiple, selective inhibitors, such as thosedescribed herein. The inhibition can be directed to inhibitligand/receptor interaction; or to inhibit expression of the ligands orreceptors; or to inhibit downstream signaling, for example. Acomposition comprising an inhibitor can be administered, or acomposition comprising a pro-drug that is metabolized into an inhibitorcan be administered; or a polynucleotide that encodes an inhibitor canbe administed in a manner that achieves expression of the encodedinhibitor in the recipient organism.

Example 10 Inhibition of Rejection of Other Organ Grafts and in OtherSpecies

The experiments described in Examples 7-9 are repeated in rodent modelsfor all other organ transplants, including kidney, liver, lung,pancreas, intestine, and esophagus; to demonstrate that therapy directedto these molecular targets for intervention is effective with respect torecipients of other organ transplants.

The experiments described in Examples 7-9, or in the precedingparagraph, are repeated in larger mammals (e.g., felines, canines,porcines, equines, bovines, primates) to demonstrate efficacy in otherspecies that may be considered more representative of humans, as aprerequisite to proving efficacy in human clinical trials.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Becausemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed to include everything within the scope ofthe appended claims and equivalents thereof. The patents, patentapplication publications and other publications (e.g., Journal articles,and web/Internet materials) referenced herein are incorporated in theirentirety.

Although the applicant(s) invented the full scope of the claims appendedhereto, the claims are not intended to encompass within their scope theprior art work of others. Therefore, in the event that statutory priorart within the scope of a claim is brought to the attention of theapplicants by a Patent Office or other entity or individual, theapplicant(s) reserve the right to exercise amendment rights underapplicable patent laws to redefine the subject matter of such a claim tospecifically exclude such statutory prior art or obvious variations ofstatutory prior art from the scope of such a claim. Variations of theinvention defined by such amended claims also are intended as aspects ofthe invention.

The patents, patent application publications and other publications(e.g., Journal articles) referenced herein are incorporated in theirentirety.

1. A method for inducing tolerance or inhibiting rejection of a cell,tissue, or organ transplant, or for inhibiting arteriosclerosis in atransplant recipient, comprising: administering to a mammaliantransplant recipient a composition that comprises an endothelial growthfactor inhibitor, in an amount effective to induce tolerance for thetransplant by the recipient, or inhibit rejection, or inhibitarteriosclerosis.
 2. A method for inducing tolerance or inhibitingrejection of a cell, tissue, or organ transplant, or for inhibitingarteriosclerosis in a transplant recipient, comprising: administering toa mammalian transplant recipient a composition that comprises an nucleicacid that comprises a nucleotide sequence that encodes an endothelialgrowth factor inhibitor, wherein the nucleic acid is expressible incells of the recipient or expressible in the transplanted cell, tissue,or organ to produce an amount of the endothelial growth factor inhibitoreffective to induce tolerance for the transplant by the recipient, orinhibit rejection, or inhibit arteriosclerosis.
 3. The method of claim2, wherein the nucleic acid further comprises at least one expressioncontrol sequence operatively connected to the sequence that encodes theendothelial growth factor inhibitor.
 4. The method of claim 2,comprising administering an expression vector that comprises the nucleicacid.
 5. The method of claim 4, wherein the vector comprises areplication deficient viral vector.
 6. The method of claim 5, whereinthe vector comprises at least one member selected from the groupconsisting of a retrovirus, an adenovirus, an adeno-associated virus, avaccinia virus and a herpesvirus.
 7. The method of claim 4, whereinexpression of the vector is inducible by administration of an exogenouspharmaceutical agent.
 8. The method of claim 4, wherein expression ofthe vector is induced by an endogenous stress in the organ transplantrecipient.
 9. The method of claim 8, wherein the stress comprises anelevation of a biological marker correlated with rejection.
 10. Themethod of claim 1, wherein the recipient is human.
 11. The method ofclaim 10, wherein the transplant is a xenograft, and the method inducestolerance for the xenograft or inhibits xenograft rejection.
 12. Themethod of claim 10, wherein the transplant is an allograft transplant,and the composition is administered in an amount effective to inducetolerance for the allograft or inhibit alloimmunity.
 13. The method ofclaim 1, wherein the composition further comprises a pharmaceuticallyacceptable carrier.
 14. The method of claim 1, where the transplant is acell or tissue transplant.
 15. The method of claim 14, wherein the cellor tissue comprises a member selected from the group consisting ofembryonic stem cells, pluripotent stem cells, hematopoietic precursorcells, neuronal precursor cells, and endothelial precursor cells. 16.The method of claim 14, wherein the cell or tissue comprises a memberselected from the group consisting of pancreatic islet cells, cardiacmyocytes, bone marrow cells, endothelial cells, and skin cells.
 17. Themethod of claim 10, wherein the transplant is an organ or organ fragmentcapable of performing functions of the organ or capable of regeneratinginto the organ.
 18. The method of claim 1, wherein the recipientreceived at least one transplanted organ, or fragment thereof, selectedfrom the group consisting of a heart, a kidney, a lung, a liver, anintestine, a pancreas, skin, and bone.
 19. The method of claim 1,wherein the recipient received at least one transplanted organ selectedfrom the group consisting of heart, lung, liver, and kidney.
 20. Themethod of claim 1, wherein the recipient received a heart transplant.21. The method of claim 1, wherein the composition is administeredlocally to the transplanted cell, tissue, or organ in the recipient. 22.The method of claim 1, wherein the composition is administeredsystemically to the recipient.
 23. The method of claim 1, wherein thecomposition is administered intravenously, intramuscularly, orintraperitoneally.
 24. The method of claim 1, wherein the composition isadministered perorally.
 25. The method of claim 1, further comprisingadministering the composition to the organ or the organ donor before thetransplant.
 26. The method of claim 1, further comprising repeatedadministration of the composition to the recipient.
 27. The method ofclaim 1, wherein the composition is administered to the recipientperioperatively, relative to the transplant operation.
 28. The method ofclaim 1, wherein the composition is administered to the recipient for1-90 days post-operatively, relative to the transplant operation. 29.The method of claim 1, wherein the composition is administered to therecipient for 1-60 days post-operatively, relative to the transplantoperation.
 30. The method of claim 1, wherein the composition isadministered to the recipient for 1-30 days post-operatively, relativeto the transplant operation.
 31. The method of claim 1, wherein thecomposition is administered to the recipient for 1-15 dayspost-operatively, relative to the transplant operation.
 32. The methodof claim 1, comprising: screening the organ transplant recipient forsymptoms of an acute rejection reaction; and administering thecomposition to the recipient upon detection of symptoms of acuterejection, in an amount effective to inhibit the rejection.
 33. Themethod of claim 1, wherein the endothelial growth factor inhibitorcomprises a compound that inhibits stimulation of at least one receptorselected from the group consisting of VEGFR-1, VEGFR-2, and VEGFR-3, bya growth factor ligand of said at least one receptor.
 34. The method ofclaim 1, wherein the endothelial growth factor inhibitor comprises acompound that inhibits stimulation of VEGFR-3 by VEGF-C or inhibitsstimulation of VEGFR-3 by VEGF-D.
 35. The method of claim 34, whereinthe compound comprises an antibody substance selected from the groupconsisting of antibody substances that immunoreact with VEGFR-3,antibody substances that immunoreact with VEGF-C, and antibodysubstances that immunoreact with VEGF-D.
 36. The method of claim 35,wherein the antibody substance is selected from the group consisting ofa humanized antibody, a human antibody, a monoclonal antibody, afragment of an antibody, and a polypeptide that comprises an antigenbinding fragment of an antibody.
 37. The method of claim 35, wherein theantibody substance is a monoclonal antibody that binds VEGFR-3 or VEGF-Cor VEGF-D and inhibits binding between VEGFR-3 and VEGF-C or -D.
 38. Themethod of claim 1, wherein the endothelial growth factor inhibitorcomprises a soluble receptor that binds to at least one endothelial cellgrowth factor.
 39. The method of claim 38, wherein the endothelialgrowth factor inhibitor comprises a soluble VEGFR-3 polypeptide thatbinds to VEGF-C or VEGF-D.
 40. The method of claim 39, wherein thesoluble VEGFR-3 polypeptide comprises the VEGFR-3 extracellular domain,or a fragment thereof sufficient to bind VEGF-C or VEGF-D.
 41. Themethod of claim 40, wherein the soluble VEGFR-3 polypeptide comprisesthe first and second immunoglobulin-like domains of the VEGFR-3.
 42. Themethod of claim 40, wherein the soluble VEGFR-3 polypeptide comprisesthe first, second, and third immunoglobulin-like domains of the VEGFR-3.43. The method of claim 38, wherein the soluble receptor is fused to animmunoglobulin constant domain.
 44. The method of claim 1, wherein theinhibitor comprises a polypeptide that comprises an amino acid sequenceat least 95% identical to amino acids 138-226 of SEQ ID NO:
 6. 45. Themethod of claim 1, wherein the inhibitor comprises a polypeptide thatcomprises an amino acid sequence at least 95% identical to amino acids47-224 of SEQ ID NO:
 6. 46. The method of claim 1, wherein the inhibitorcomprises a polypeptide that comprises an amino acid sequence at least95% identical to amino acids 47-314 of SEQ ID NO:
 6. 47. The method ofclaim 1, wherein the inhibitor comprises a polypeptide that comprises anamino acid sequence at least 95% identical to amino acids 24-775 of SEQID NO: 6 or fragments thereof that bind VEGF-C.
 48. The method of claim1, wherein the inhibitor comprises an antisense nucleic acid or aninterfering RNA nucleic acid that inhibits expression of an endothelialcell growth factor or endothelial cell growth factor receptor.
 49. Themethod of claim 48, wherein the inhibitor is a short interfering RNAthat inhibits expression of a protein selected from the group consistingof VEGFR-3, VEGF-C, and VEGF-D.
 50. The method of claim 48, wherein theinhibitor is an antisense nucleic acid that inhibits expression of aprotein selected from the group consisting of VEGFR-3, VEGF-C, andVEGF-D.
 51. The method of claim 34, further comprising administering tothe recipient a composition that comprises at least one growth factorinhibitor selected from the group consisting of: inhibitors of VEGFR-1with one or more of its ligands; inhibitors of VEGFR-2 with one or moreof its ligands; inhibitors of PDFFR-alpha with one or more of itsligands; inhibitors of PDGFR-beta with one or more of its ligands;wherein the combination of inhibitors are administered in amountseffective to induce tolerance for the transplant by the recipient, orinhibit rejection, or inhibit arteriosclerosis.
 52. The method of claim33, wherein the compound comprises bevacizumab (Avastin®) or Ranibizumab(Lucentis®).
 53. The method of claim 33, wherein the compound is amultivalent inhibitor of two or more receptors selected from the groupconsisting of VEGFR-1, VEGFR-2, and VEGFR-3.
 54. The method of claim 53,wherein the multivalent compound inhibits VEGFR-3 and at least onereceptor selected from VEGFR-1 and VEGFR-2.
 55. The method of claim 1,comprising administering to the recipient a composition that inhibitsligand binding to VEGFR-2 and inhibits ligand binding to VEGFR-3. 56.The method of claim 1, further comprising administering animmunosuppressive agent to the organ transplant recipient.
 57. Themethod of claim 56, wherein the immunosuppressive agent comprises atleast one agent selected from the group consisting of corticosteriods,calcineurine inhibitors, antiproliferative agents, monoclonalantilymphocyte antibodies, and polyclonal antilymphocyte antibodies. 58.The method of claim 56, wherein the immunosuppressive agent comprises atleast one compound selected from the group consisting of: Tacrolimus,Mycophenolic acid, Prednisone, Ciclosporin, Azathioprine, Basiliximab,Cyclosporine, Daclizumab, Muromonab-CD3, Mycophenolate Mofetil,Sirolimus, Methylprednisolone, Atgam, Thymoglobulin, OKT3, Rapamycin,Azathioprine, Cyclosporine, and Interleukin-2 Receptor Antagonist. 59.The method of claim 1, further comprising administering an antibiotic orantifungal agent to the recipient.
 60. The method of claim 1, furthercomprising administering to a donor organism a composition thatcomprises an endothelial growth factor inhibitor, prior to harvesting acell, tissue, or organ for transplantation into the recipient.
 61. Themethod of claim 1, further comprising contacting a cell, tissue, ororgan with a composition that comprises an endothelial growth factorinhibitor, prior to transplanting the cell, tissue, or organ into themammalian organ transplant recipient.
 62. The method of claim 1, furthercomprising administering to a donor organism, prior to harvesting cells,tissue, or an organ for transplantation, a composition that comprises annucleic acid that comprises a nucleotide sequence that encodes anendothelial growth factor inhibitor, wherein the nucleic acid isexpressible in cells of the tissue or organ to be transplanted.
 63. Themethod of claim 1, further comprising contacting a cell, tissue, ororgan with a composition that comprises an nucleic acid that comprises anucleotide sequence that encodes an endothelial growth factor inhibitor,prior to transplanting the cell, tissue, or organ into the recipient.64. A method of preparing a donor cell, tissue, or organ for allograftor xenograft transplantation comprising contacting the cell, tissue, ororgan with a composition that comprises an endothelial growth factorinhibitor, prior to transplanting the cell, tissue, or organ into amammalian organ transplant recipient.
 65. A method of preparing a donorcell, tissue, or organ for allograft or xenograft transplantationcomprising contacting the cell, tissue, or organ with a composition thatcomprises an nucleic acid that comprises a nucleotide sequence thatencodes an endothelial growth factor inhibitor, prior to transplantingthe cell, tissue, or organ into a mammalian organ transplant recipient.66. A composition that comprises an endothelial growth factor inhibitor,an immunosuppressant, and a pharmaceutically acceptable carrier.
 67. Thecomposition according to claim 66, wherein the inhibitor and theimmunosuppressant are present in the composition in synergisticallyeffective amounts.
 68. The method of claim 2, comprising: screening theorgan transplant recipient for symptoms of an acute rejection reaction;and administering the composition to the recipient upon detection ofsymptoms of acute rejection, in an amount effective to inhibit therejection.
 69. The method of claim 56, further comprising administeringan antibiotic or antifungal agent to the recipient.
 70. The method ofclaim 56, further comprising administering to a donor organism acomposition that comprises an endothelial growth factor inhibitor, priorto harvesting a cell, tissue, or organ for transplantation into therecipient.
 71. The method of claim 69, further comprising administeringto a donor organism a composition that comprises an endothelial growthfactor inhibitor, prior to harvesting a cell, tissue, or organ fortransplantation into the recipient.